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PRINCIPLES OF
SOIL MICROBIOLOGY

PRINCIPLES OF
SOIL MICROBIOLOGY

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SELMAN A. WAKSMAN

Associate Professor of Soil Microbiology,
Rutgers University, and Microbiologist of the
New Jersey Agricultural Experiment Stations

LONDON

BAILLIERE, TINDALL AND COX

8 HENRIETTA STREET, COVENT GARDEN, W. C. 2

1927

ALL RIGHTS RESERVED, 1927

PRINTED IN AMERICA

COMPOSED AND PRINTED AT THE

WAVERLY PRESS

BALTIMORE, MD., U. 8. A.

In the years of their seventy-fifth and seventieth

anniversaries respectively, this book is dedicated

to Professors

M. W. BEIJERINCK

and
S. WINOGRADSKY

the investigators who have thrown the first UgJd
upon some of the most important soil proc-
esses and whose contributions can
well be considered first and fore-
most in the science of Soil
Microbiology

PREFACE

Although the biochemical processes in the soil as well as the nature
of the microorganisms present there have received considerable atten-
tion from various points of view and although an extensive literature has
accumulated, not only dealing with soil processes in general but even
with certain specific activities of the organisms, our present knowledge
of the soil microflora and microfauna and of the numerous transforma-
tions that they bring about has not advanced beyond a mere beginning
of a systematic study. The isolation of numerous microorganisms from
the soil, their identification and cultivation upon artificial media is
very important but such data do not tell what role they play in the soil.
A knowledge of the activities of certain organisms isolated from the soil
is certainly necessary, but that is not a knowledge of the extent to
which these processes take place in the soil itself. A book on soil
microbiology should include a study of the occurrence of microorganisms
in the soil, their activities and their role in soil processes. It is this
last phase which has been studied least and where the information
available is far from satisfactory in explaining what is taking place in
the soil. This is due largely to the limitations of the subject which
depends for its advance on botany, zoology, bacteriology, chemistry,
including biological and physical, and especially upon the advance of
our understanding of the physical and chemical conditions of the soil.

There are various kinds of audiences to which a book on soil micro-
biology may appeal. There is the scientific farmer who may search for
a better understanding of the processes taking place in the soil, those
processes which control the growth of his crops and indirectly influence
the growth of his animals. There is the agronomist, who is interested
in the fundamental reactions controlling soil fertility, by reason of the
need of directing such processes towards a greater utilization of the
nutrients added to the soil or stored away in the soil organic matter.
There is the investigator, the soil chemist or the soil microbiologist, who,
in attacking problems dealing with the occurrence of microorganisms
in the soil, their activities, and especially with the relation of these
activities to the physical and chemical soil conditions, seeks for specific
or general information. These investigators may deal with organisms

Vlll PREFACE

or processes which could be better understood when correlated with the
other soil organisms and the numerous other processes. An attempt
has been made to compile a book which will be of service not only to the
investigators in soil science, but also to workers in allied sciences,
especially botany, plant physiology, plant pathology and bacteriology,
as well as to the general student in agriculture.

This book is a collection of known facts concerning microorganisms
found in the soil and their activities; it is a study of the literature dealing
with the science in question; it is an interpretation of the facts already
presented; it indicates the various lines of investigation and notes where
further information is especially wanted. Soil microbiology is a science
which is at the very base of our understanding of agricultural processes
and the practice of agriculture; it comprises a number of sciences. The
book may, therefore, be looked upon more as an introduction to further
research rather than as an ordinary text-book; as of help to those work-
ing in the allied sciences, who are desirous of obtaining some information
concerning the soil population and its activities.

If this volume will help to disclose to the reader some of the numerous
interrelated processes in the soil, if it will present in a clearer light to the
chemist, the physiologist, the botanist, the bacteriologist and the
zoologist the nature of the many scientific and practical problems
awaiting the investigator, if it contributes in a small measure toward
making soil science an exact science, the author will feel that he has been
amply rewarded.

The author is greatly indebted to his various colleagues for reading
and criticizing the different chapters of the book and for the many
helpful suggestions generously offered, especially to Dr. H. J. Conn, of
the New York Agricultural Experiment Station, for reading Chapters
I and VI; to Dr. B. M. Bristol Roach, of the Rothamsted Experimental
Station, and Dr. G. T. Moore, of the Missouri Botanical Garden, for
reading the Chapter on Algae; to Dr. Ch. Thorn of the Bureau of
Chemistry, for reading the Chapter on Fungi; to Dr. M. C. Rayner, of
Bedford College, London, for reading the section dealing with Mycor-
rhiza Fungi; to Dr. A. T. Henrici, of the University of Minnesota,
for reading the Chapter on Actinomyces; to Dr. W. M. Gibbs, of the
Idaho Agricultural Experiment Station, for reading the Chapter on
Nitrifying Bacteria; to Dr. A. L. Whiting, of the University of Wis-
consin and to Dr. L. T. Leonard of the Bureau of Plant Industry, for
reading the Chapter on Nodule Bacteria; to Dr. R. Burri, of Liebefeld,
Switzerland, and to Dr. I. C. Hall, of the Colorado Medical School,

PREFACE IX

for reading the Chapter on Anaerobic Bacteria; to Mr. D. W. Cutler,
Mr. H. Sandon, of the Rothamsted Experimental Station, and Prof.
C. A. Kofoid, of the University of California, for reading the Chapter on
Protozoa; to Dr. N. Cobb and Dr. Steiner, of the Bureau of Plant
Industry, for reading the Chapter on Soil Invertebrates; to Dr. O.
Meyerhof, of the K. Wilhelm Institute, Berlin, for reading the Chapter
on Energy Transformation; to Dr. T. B. Osborne, of the Connecticut
Agricultural Experiment Station, for reading the Chapter on Protein
Transformation; to Mr. A. Bonazzi, of Cuba, for reading the Chapter on
Non-symbiotic Nitrogen Fixation; to Dr. R. L. Jones, of the University
of Wisconsin, for reading Chapter XXX; to Dr. E. B. Fred, of the Uni-
versity of Wisconsin, for reading the Chapter on Nitrate-reducing
Bacteria; to Prof. D. R. Hoagland and Dr. W. P. Kelley, of the Uni-
versity of California for reading Chapters 24 and 25 respectively; to
the members of the Soil Microbiology Division of the New Jersey Ag-
ricultural Experiment Station, especially to Dr. J. G. Lipman and
Dr. R. L. Starkey, for reading various parts of the book, and to all
those who have generously allowed the use and reproduction of the
various illustrations in the text.

Selman A. Waksman.

August 25, 1826.

New Brunswick., N. J., U. S. A.

A CLASSIFIED LIST OF BOOKS FOR REFERENCE IN SOIL

MICROBIOLOGY

CLASSIFICATION OF ORGANISMS

Bacteria

Bergey, D. H. A manual of determinative bacteriology. Williams & Wilkins

Co., Baltimore. 1923.
Buchanan, R. E. General systematic bacteriology. History, nomenclature,

groups of bacteria. (Monogr. on systematic bacteriology, vol. 1.)

Williams & Wilkins Co., Baltimore. 1925.
Chester, F. D. A manual of determinative bacteriology. Macmillan Co.,

New York and London. 1901.
FlUgge, C. Die Mikroorganismen. 2 vols., 3rd Ed. Leipzig. 1896.
Lehmann, K. B., and Neumann, R. O. Atlas und Grundrisz der Bakteriologie.

6th Ed. Teil. I, Atlas. Teil II, Text. J. F. Lehmann. Miinchen.
• 1920.
Matzuschita, T. Bakteriologische Diagnostik. Jena. 1902.
Migula, W. System der Bakterien. Jena. Bd. I, 1897; Bd. II. 1900.
Winslow, C. E. A., and Winslow, A. R. The systematic relationships of the

Coccaceae. J. Wiley & Sons, New York. 1908.

Fungi

Brefeld, O. Botanische Untersuchungen iiber Schimmelpilze. 1872.
Clements, F. E. The genera of fungi. Minneapolis. 1909.
Coupin, H. Fungi (champignons). Album Gen. Cryptogames. 1921.
DeBary, A. Comparative morphology and biology of the fungi, mycetozoa

and bacteria. (Tr. Gainey, H. E. F. and Balfour, I. B.) Clarendon

Press, Oxford. 1887.
Engler, A., and Prantl, K. A. Die natiirlichen Pflanzenfamilien. Teil I,

Abt. I. Engelmann, Leipzig. 1900.
Fischer, E. Pilze. Handwort. Naturwiss. V. 7. Jena. 1912.
Gaumann, E. Vergleichende Morphologie der Pilze. G. Fischer, Jena. 1926.
Lindau, G. Fungi imperfecti. Hyphomycetes. Rabenhorst's Kryptogamen

Flora. Vols. 8 and 9. 1907-1910.
Lindau, G. K. Kryptogamenflora fur Anfanger. 2 (1) Die mikroskopischen

Pilze (Myxomyceten, Phycomyceten und Ascomyceten). 2nd. Ed.

J. Springer, Berlin. 1922.
Saccardo, P. A. Sylloge Fungorum. Patavia. 1882-1913.
Wettstein, R. Handbuchder systematischen Botanik. 3d Ed. Vol.1. Wien.

1923.
Zopf, W. Die Pilze in morphologischer, physiologischer, biologischer und

systematischer Beziehung. Breslau. 1890.

Xll CLASSIFIED LIST OF BOOKS

Algae

Chodat, R. Monographie d'Algues en culture pure. Bern. 1913.

Cotjpin, H. Les algues du globe. V. 1. Paris. 1912.

Engler, A., and Prantl, K. A. Die naturlichen Pflanzenfamilien. Teil I,

Abt. la. Leipzig. 1897.
Heurck, H. J. Traite des Diatom6es. Anvers. 1899.
Lindau, G. Kryptogamenflora fur Anfanger. Bd. IV, 1, 2 and 3. Die Algen.

J. Springer, Berlin. 1914-1916.
Oltmanns, Fr. Morphologie und Biologie der Algen. 3 vols. 2nd Ed. G.

Fischer, Jena. 1922.
Pascher, A. Die Susswasserflora Deutschlands, Osterreichs und der Schweiz.

Jena. H. 4 to H. 12. G. Fischer. 1915-1925.
Tilden, J. E. Minnesota algae. Minneapolis. 1910.
de Toni, G. B. Sylloge algarum omnium hucus que cognitarum. 1889-1907.

Padua.
West, G. S. Algae. Cambridge Botanical Handbooks. I. Cambr. Univ.

Press. 1916.

Yeasts

Chapman, A. C, and Baker, F. G. C. An atlas of the saccharomycetes. Lon-
don. 1906.

Jorgensen, A. Die Mikroorganismen der Giirungsgewerbe. 5th Ed.

Guillermond, A. The yeasts. (Trans. F. W. Tanner.) J. Wiley & Sons, New
York. 1920.

Henneberg, W. Garungsphysiologisches Praktikum. Berlin. 1909.

Klocker, A. Die Giirungsorganismen in der Theorie und Praxis der Alkohol-
garungsgewerbe. Max Waag. 2nd Ed. Stuttgart. 1906.

Kohl, F. G. Die Hefepilze. Quelle und Meyer. Leipzig. 1908.

Lindner, A. Saccharomycetineae. In Kryptogamenflora der Mark Branden-
burg. Bd. 7, H. 1. Leipzig. 1905.

Protozoa

Butschli, O. Protozoa. In Bronn's Thierreich. 1882-1887.

Calkins, G. N. The biology of the protozoa. Lea & Febiger, Philadelphia.

1926.
Cash, J. The British freshwater Rhizopods and Heliozoa. Roy. Soc. London.

1905-1921.
Doflein, F. Lehrbuch der Protozoenkunde. 4th Ed. Jena. G. Fischer.

1916.
Lister, A. A monograph of the mycetozoa. 3rd Ed. Rev. G. Lister, Brit.

Museum, London. 1925.
MacBride, T. H. North American slime molds. 2nd Ed. Macmillan. 1922.
Minchin, E. A. An introduction to the study of the protozoa. E. Arnold,

London. 1912.
Pascher, A., and Lemmermann, E. Die Susswasserflora Deutschlands, Oster-
reichs und der Schweiz. H. 1 to H. 3. G. Fischer, Jena. 1913.
Wenyon, C. M. Protozoology. 2 vols. Bailliere, Tindall and Cox. London.

1926.

CLASSIFIED LIST OF BOOKS Xlll

Schaeffer, A. A. Taxonomy of the Amebas with description of thirty-nine
new marine and fresh-water species. Vol. 24, Carnegie Inst. Wash.,
Dept. Marine Biol. 1926.

Nematodes

Baylis, H. A., and Daubney, R. A synopsis of the families and genera of

Nematoda. Brit. Museum, London. 1926.
De Man, J. G. Nouvelles recherches sur les nematodes libres terricoles. M.

Nijhoff, Hague. 1922.
Micoletzky, H. Die freilebenden Erd-Nematoden. Arch. Naturges. 87

(Abt. A). 1922.
Yorke, W., and Maplestone, P. A. The nematode parasites of vertebrates.

P. Blakiston's Son & Co., Philadelphia. 1926.
Ward, H., and Whipple, G. C. Fresh Water Biology. J. Wiley & Sons, New

York. 1918.

THEORETICAL AND APPLIED MICROBIOLOGY

General Microbiology

Baumgartel, T. Grundriss der theoretischen Bakteriologie. J. Springer,
Berlin. 1924.

Benecke, W. Bau und Leben der Bakterien. Teubner, Leipzig. 1912.

Conn, H. W., and Conn, H. J. Bacteriology. Williams & Wilkins Co., Balti-
more. 1923.

Ellis, D. Practical bacteriology. London. 1923.

Fischer, A. Vorlesungen iiber Bakterien. Jena. 1903.

Hiss, P. H., and Zinsser, H. A text book of bacteriology. 5th Ed. Appleton
& Co., New York. 1922.

Jordan, E. O. A text-book of general bacteriology. 7th Ed. Philadelphia,
1922.

Kendall, A. J. Bacteriology, general, pathological and intestinal. 2d Ed.
Philadelphia. 1921.

Kolle, W., and Wassermann, A. Handbuch der pathogenen Mikroorganismen.
2d Ed. Jena. 1913.

Kruse, W. Allgemeine Mikrobiologie. Vogel, Leipzig. 1910.

Kruse, W. Einfiihrung in die Bakteriologie. W. de Gruyter, Berlin. 1920.

Meyer, A. Die Zelle der Bakterien. Jena. 1912.

Omeliansky, W. L. Principles of microbiology (Russian). U. S. S. R.
Leningrad. 5th Ed. 1924.

Park, W. H., Williams, A. W. and Krumwiede, C. Pathogenic microorgan-
isms. Eighth Ed. Lea & Febiger, Philadelphia. 1924.

Agricultural Microbiology

Baumgartel, T. Vorlesungen iiber landwirtschaftliche Mikrobiologie. P.

Parey, Berlin. 1925-1926.
Buchanan, R. E. Agricultural and industrial bacteriology. New York. 1922.
Chudiakov, H. H. Agricultural Microbiology (Russian). Moskau. 1926.

XIV CLASSIFIED LIST OF BOOKS

Dcclaux, E. Traits de Microbiologic. Masson et Cie, Paris. Vols. I-IV.

1898-1901.
Fuhrmann, F. Vorlesungen Uber technische Mykologie. G. Fischer, Jena.

1913.
Gkeaves, J. E. Agricultural bacteriology. Lea and Febiger, Philadelphia.

1922.
Janke, A. Allgemeine technische Mikrobiologie. I. Steinkopff. Dresden and

Leipzig. 1924.
Kayser, E. Microbiologic appliquee a la fertilisation du sol. J. B. Bailliere,

Paris. 1921.
Kossowicz, A. Einfiihrung in die Agrikulturmykologie. Teil I, Bodenbak-

teriologie. Borntraeger, Berlin. 1912.
Lafar, F. Handbuch der technischen Mykologie. 5 Vols. G. Fischer, Jena.

1904-1913.
Lipman, J. G. Bacteria in relation to country life. The Macmillan Co., New

York. 1911.
Lohnis, F. Handbuch der landwirtschaftlichen Bakteriologie. Borntraeger,

Berlin. 1910.
Lohnis, F., and Fred, E. B. Agricultural bacteriology. McGraw-Hill, New

York. 1923.
Russell, Sir John, and others. The microorganisms of the soil. Long-
mans, Green & Co., London. 1923.
Marshall, C. E. Microbiology. Blakiston, Philadelphia. 3rd Ed. 1922.
Russell, H. L., and Hastings, E. G. Agricultural bacteriology. 1915.
Rossi, G. de. Microbiologia agraria e technica. Unione Tip, Torino. 1921—

1926.
Smith, E. F. Bacteria in relation to plant diseases. Vol. I, 1905; vol. II, 1911;

Vol. Ill, 1914.
Stoklasa, J., and Doerell, E. G. Handbuch der physikalischen und biochem-

ischen Durchforschung des Bodens. P. Parey, Berlin. 1926.
Tanner, F. W. Bacteriology and mycology of foods. New York. 1919.

Manuals of Bacteriologic Technic

Abderhalden, E. Handbuch der biologischen Arbeitsmethoden. Abt. XI.

2nd Ed. 1924-1926.
Abel, R. Bakteriologisches Taschenbuch. C. Kabitzsch, Leipzig. 26th Ed.

1923.
American Public Health Association standard methods for the examination of

water and sewage. 1915.
Barnard, J. E., and Welch, F. V. Practical photo-micrography. 2nd Ed.

Longmans, Green & Co., New York. 1925.
Besson, A. Technique microbiologique et s6rothe>apique. 3 vol. 1921-1923.
Burgess, P. S. Soil bacteriology laboratory manual. 1914.
Emich, F. Lehbach der Mikrochemie, Munich. 1926.
Ehringhaus, A. Das Mikroskop, seine wissenschaftlichen Grundlagen und

seine Anwendung. Teubner, Leipzig.
Ehrlich and Weigert. Encyclopedic der mikroscopischen Technik. Vol. I

& II. 1910.

CLASSIFIED LIST OF BOOKS XV

Eyre, J. W. H. Bacteriological technique. 2nd Ed. 1913.

Fred, E. B. A laboratory manual of soil bacteriology. Blakiston Co., Phila-
delphia. 1916.

Gage, S. H. The microscope. 14th Ed. Comstock, Ithaca, N. Y. 1925.

Giltner, W. Laboratory manual in general microbiology. 3rd Ed. J. Wiley
and Sons, New York. 1926.

Hager, H. Das Mikroskop und seine Anwendung. 13th Ed. by F. Tobler.
J. Springer, Berlin. 1925.

Heinemann, P. G. A laboratory guide in bacteriology. 3rd Ed. 1915.

Hewlett, R. T. A manual of bacteriology. London. 1921.

Koch, A. Mikrobiologisches Praktikum. J. Springer, Berlin. 1922.

Kraus, R., and Uhlenhuth, P. Handbuch der mikrobiologischen Technik.
3 vols. Urban and Schwarzenberg, Berlin. 1923-1924.

Kuster, E. Kultur der Mikroorganismen. 3rd Ed. Teubner, Leipzig. 1921.

Langeron, M. Precis de microscopie. 4th Ed. Masson et Cie, Paris. 1925.

Laubenheimer. Lehrbuch der Mikrophotographie. 1920.

Lee, A. B. The microtomist's Vade-Mecum. 8th Ed. Churchill, London.
1921.

Lipman, J. G., and Brown, P. E. Laboratory guide in soil bacteriology. 1911.

Lohnis, F. Landwirtschaftlich-bakteriologisches Praktikum. 2nd Ed. Born-
traeger, Berlin. 1920.

Mace, E. Traite pratique de bact6riologie. Atlas de Microbiologic Bailliere.
Paris. 1913.

Meyer, A. Praktikum der botanischen Bakterienkunde. G. Fischer, Jena.
1903.

Prowazek, S. V. (V. Jollos). Taschenbuch der mikroskopischen Technik der
Protistenuntersuchung. 3rd Ed. J. A. Barth, Leipzig. 1922.

Sieben, H. Einfuhrung in die botanische Mikrotechnik. Fischer, Jena. 1913.

Schneider, A. Bacteriological methods in food and drug laboratories. Blakis-
ton, Philadelphia. 1915.

SOILS AND PLANTS

The Physics and Chemistry of Soils and Manures

Airman, C. M. Manures and principles of manuring. London. 1910.
Cameron, F. K. The soil solution, the nutrient medium for plant growth.

Easton, Pa. 1911.
Clarke, F. W. The data on geochemistry. Bui. 770, U. S. Geol. Survey. 1924.
Ehrenberg, P. Die Bodenkolloide. 3rd Ed. Steinkopff, Dresden and Leipzig.

1920.
Emerson, F. V. Agricultural geology. J. Wiley & Sons, New York. 1920.
Fraps, G. S. Principles of agricultural chemistry. The Chemical Publishing

Co., Easton, Pa. 1913.
Glinka, K. D. Soils of Russia and adjoining countries (Russian). Gosizdat,

Moskau. 1923.
Hall, A. D. The soil, an introduction to the scientific study of the growth of

crops. 3rd Ed. London. 1920.
Hinkle, S. F. Fertility and crop production. Sandusky, Ohio. 1925.

XVI CLASSIFIED LIST OF BOOKS

Lyon, T. L., and Buckman, H. C. The nature and properties of soils. Mac-

millan, New York. 1922.
Heiden, E. Lehrbuch der Dungerlehre. 2 parts. Hannover. 1879-1887.
Hilgard, E. W. Soils, their formation, properties, composition and relations

to climate and plant growth. Macmillan, New York. 1912.
Hoering, P. Moornutzung und Torfverwertung mit besonderer Berucksichti-

gung der Trockendestillation. J. Springer, Berlin. 1915.
Honcamp, F., and Nolte, O. Agrikulturchemie. T. Steinkopff, Dresden and

Leipzig. 1924.
Hopkins, C. G. Soil fertility and permanent agriculture. Ginn & Co., Boston.

1910.
Kober, L. Der Bau der Erde. Borntraeger, Berlin. 1921.
Mayer, A. Die Dungerlehre. 7th Ed. C. Winters, Heidelberg. 1924.
Merrill, G. P. Rocks, rock weathering and soils. Macmillan, New York.

1897.
Mitscherlich, E. A. Bodenkunde fur Land- und Forstwirte. 3rd Ed. P.

Parey, Berlin. 1920.
Murray, J. A. The science of soils and manures. 3rd Ed. D. VanNostrand Co.

1925.
Puchner, H. Der Torf. F. Enke, Stuttgart. 1920.
Ramann, E. Bodenkunde. 3rd Ed. J. Springer, Berlin. 1911.
Russell, J. Soil conditions and plant growth. Longmans, Green & Co. 4th

Ed. London. 1921.
Van Slyke, L. L. Fertilizers and crops. O. Jodd Co., New York. 1912.
Warington, R. Lectures on some of the physical properties of soil. Oxford.

1900.
Wheeler, H. J. Manures and fertilizers. New York. 1913.
Wiley, H. W. Principles and practice of agricultural analysis. Vol. I. Soils.

3rd Ed. Chemical Publ. Co., Easton, Pa. 1926.

The Soil Environment and Higher Plants

Brenchley, W. E. Inorganic plant poisons and stimulants. Cambridge. 1914.
Clements, F. E. Aeration and air content; the role of oxygen in root activity.

Carnegie Inst. Wash. Publ. No. 315. 1921.
Czapek, F. Biochemie der Pflanzen. 2te Aufl. 3 vols. Jena, vol. 1, 1913;

vol.2, 1920; vol. 3, 1921.
Hahn, J. Handbuch der Klimatologie. 3 vols. Stuttgart. 1908-1911.
Jost, L. Plant physiology. Tr. R. J. H. Gibson. Oxford. 1907-1913.
Kolkwitz, R. Pflanzenphysiologie. G. Fischer, Jena. 1922.
Kostytschew, S. Pflanzenatmung. J. Springer, Berlin. 1925.
Lundegardh, H. Klima und Boden in ihrer Wirkung auf das Pflanzenleben.

G. Fischer, Jena. 1925.
Palladin, V. I. Plant physiology. Trans, by B. E. Livingston. 2nd Ed.

Blakiston, Philadelphia. 1923.
Pfeffer, W. The physiology of plants, a treatise upon the metabolism and

sources of energy in plants. Tr. A. J. Ewart. 3 vols. Oxford. 1900-

1906.

CLASSIFIED LIST OF BOOKS XV11

TREATISES IN GENERAL SCIENCES
General Biology, Physiology and Physiological Chemistry

Abderhalden, E. Handbuch der biologischen Arbeitsmethoden. 2nd Ed.

Urban & Schwarzenberg, Berlin. 1920-1926.
Abderhalden, E. Biochemiscb.es Handlexikon. 11 vols. Berlin. 1911-1924.
Bayliss, W. M. Principles of general physiology. 4th Ed. London. 1924.
Bayliss, W. M. The nature of enzyme action. London. 1925.
Cohnheim, O. Enzymes. 1912.
Effront, J. Enzymes and their applications. Trans. S. C. Prescott, New York.

1902. Biochemical catalysts in life and industry. New York. 1917.
Euler, H. Chemie der Enzyme. 2nd Ed. Bergmann, Munchen. 2 vols.

1922-1924.
Etjler, H. Grundlagen und Ergebnisse der Pflanzenchemie, nach der schwedis-

chen Ausgabe bearbeitet. I Teil, Das chemische Material der Pfian-

zen. Braunschweig. 1908. II Teil, Die allgemeinen Gesetze des

Pflanzenlebens. Ill Teil, Die chemischen Vorgange im Pflanzen-

korper. Braunschweig. 1909.
Fowler, G. J. Bacteriological and enzyme chemistry. Longmans, Green &

Co., New York. 1911.
Haas, P., and Hill, T. G. An introduction to the chemistry of plant products.

3rd Ed. London. 1921.
Hartmann, M. Allgemeine Biologie. Jena. 1925.
Henry, T. A. The plant alkaloids. Philadelphia. 1913.
Hober, R. Physikalische Chemie der Zelle und der Gewebe. 5th Ed. Leipzig

and Berlin. 1924.
Loeb, J. The dynamics of living matter. New York. 1906.
Loeb, J. The mechanistic conception of life: biological essays. Chicago.

1912.
Loeb, J. The organism as a whole, from the physicochemical viewpoint. New

York and London. 1916.
Loeb, J. Proteins and the theory of colloidal behavior. 2nd Ed. McGraw-Hill,

New York. 1924.
Mathews, A. P. Physiological chemistry. 3rd Ed. New York. 1920.
Oppenheimer, C. Handbuch der Biochemie der Menschen und der Tiere. 2nd

Ed., 5 vols. G. Fischer, Jena. 1924-1926.
Oppenheimer, C. Die Fermente und ihre Wirkungen. 5th Ed., 2 vols. G.

Thieme, Leipzig. 1925-1926.
Robertson, T. B. Principles of biochemistry. Lea & Febiger, Philadelphia.

1920.
Robertson, T. B. Physical chemistry of the proteins. Longmans, Green & Co.,

New York and London. 1918.
Schorger, A. W. Chemistry of cellulose and wood. McGraw-Hill, New York.

1926.
Thatcher, R. W. The chemistry of plant life. McGraw-Hill, New York. 1924.
Verworn, M. Allgemeine Physiologie, ein Grundriss der Lehre vom Leben.

6th Ed. Jena. 1915.

XV111 CLASSIFIED LIST OF BOOKS

Wiesner, J. V. Die Rohstoffe des Pflanzenreiches. 3rd Ed., 3 vols., Engel-

mann, Leipzig. 1921.
Wohlgemuth, J. Grundrisz der Fermentmethoden. Berlin. 1913.

Physics and Chemistry, as applied to Biology

Bechhold, H. Colloids in Biology and Medicine. New York. 1919. (Tr.

J. G. M. Bullowa.)
Clark, W.M. The determination of hydrogenions. 2ndEd., Williams & Wilkins

Co., Baltimore. 1922.
Cohen, E. Physical chemistry for physicians and biologists. (Tr. M. Fischer.)

New York. 1903.
Eichwald, E., and Foder, A. Die physikalisch-chemischen Grundlagen der

Biologic Berlin. 1919.
Findlay, A. Osmotic pressure. 2nd Ed. London. 1919.
Freundlich, H. Kapillarchemie, eine Darstellung der Chemie der Kolloide und

verwandter Gebiete. Leipzig. 1923.
Hatschek, E. An introduction to the physics and chemistry of colloids. 4th Ed.,

London and Philadelphia. 1922.
Hedin, S. G. Grundziige der physikalischen Chemie in ihrer Beziehung zur

Biologic J. F. Bergmann, Munchen. 1924.
Jellinek, K. Lehrbuch der physikalischen Chemie. 2 vols. Stuttgart. 1914—

1915.
Kolthoff, I. M., and Furman, N. H. Indicators. J. Wiley & Sons, New York.

1926.
Lewis, G. N., and Randall, M. Thermodynamics and the free energy of chem-
ical substances. McGraw-Hill, New York. 1923.
Lewis, W. C. McC. A system of physical chemistry. 3 vols., 3d and 4th Ed.

Longmans, Green & Co., London and New York. 1920-1925.
Lotka, A. J. Elements of physical biology. Williams & Wilkins Co., Baltimore,

Md. 1926.
McClendon, J. F., and Medes, G. Physical chemistry in medicine. W. B.

Saunders Co., Philadelphia. 425 p. 1925.
Nernst, W. Theoretische Chemie vom Standpunkte der Avogadroschen Regel

und der Thermodynamik. 10th Ed., Enge. Stuttgart. 1921.
Philip, J. C. Physical chemistry: its bearing on biology and medicine. Long-
mans, Green & Co., New York and London. 3d Ed. 1925.
Taylor, W. W. The chemistry of colloids and some technical applications. 3d

Ed. London. 1921.
Waksman, S. A., and Davison, W. C. Enzymes. Williams & Wilkins Co.,

Baltimore. 1926.
Washburn, E. W. An introduction to the principles of physical chemistry

from the standpoint of modern atomistics and thermodynamics. 2d

Ed. New York. 1921.
Willows, R. S., and Hatchek, E. Surface tension and surface energy and their

influence on chemical phenomena. 3d Ed. London. 1923.
Zsigmondy, R., Spear, E. B., and Norton, J. F. The chemistry of colloids.

New York. 1917.

CLASSIFIED LIST OF BOOKS XIX

Mathematics

Davenport, C. B. Statistical methods, with special reference to biological

variation. 3d Ed. New York. 1914.
Fischer, R. A. Statistical methods for research workers. Oliver and Boyd,

Edinburgh. 1925.
Mellor, W. J. Higher mathematics for students of chemistry and physics, with

special reference to practical work. 4th Ed. London. 1913.
Nernst, W., and Schoenflies, A. Einfiihrung in die mathematische Behand-

lung der Naturwissenschaften. Berlin. 1919.

CONTENTS

PART A. OCCURRENCE AND DIFFERENTIATION OF MICRO-
ORGANISMS IN THE SOIL

Chapter I

NUMBERS OF DIFFERENT GROUPS OF MICROORGANISMS FOUND IN THE SOIL AND
METHOD OF DETERMINATION

The occurrence of microorganisms in the soil. Proof of microbial activities
in the soil. Methods of study. Direct microscopic method. Organisms
found in the soil by the direct microscopic method. Cultural methods for
demonstrating the kinds of organisms active in the soil. Cultural methods
for the determination of numbers of microorganisms in the soil. Culture
media. Sampling of soil. Treatment of soil samples and preparation of
plates. Incubation of plates and counting of organisms. Mathematical
interpretation of results. Comparison of plate and microscopic methods.
Numbers of bacteria in the soil. Bacterial numbers in manure. Numbers
of bacteria in the soil during different seasons of the year. Distribution
of bacteria at various depths. Numbers of specific physiological groups of
bacteria. Numbers of actinomyces in the soil. Numbers of fungi in the
soil. Methods of counting protozoa. Numbers of protozoa in the soil. .. . 3

PART B. ISOLATION, IDENTIFICATION, AND CULTIVATION OF
SOIL MICROORGANISMS

Chapter II

PURE CULTURE STUDY AND CLASSIFICATION OF SOIL BACTERIA

Pure culture study. Differentiating characters of bacteria. Life cycles of
bacteria. Classification of soil bacteria based upon their physiological
activities 53

Chapter III

AUTOTROPHIC BACTERIA

The nature of autotrophic bacteria. Bacteria deriving their energy from
nitrogen compounds. Solid media for the isolation and cultivation of the
nitrite forming organisms. Morphology of the nitrite forming organisms.
Nitrate forming organisms (Nitrobacter). Occurrence of nitrifying bac-
teria in the soil. Bacteria deriving their energy from the oxidation of
sulfur and its compounds. Classification of sulfur bacteria. Oxidation of
selenium and its compounds. Bacteria oxidizing iron compounds. Bac-

3038

XX11 CONTENTS

teria obtaining their energy from the oxidation of simple carbon com-
pounds. Methane bacteria. Bacteria oxidizing carbon monoxide. Bac-
teria oxidizing hydrogen 61

Chapter IV

BACTERIA FIXING ATMOSPHERIC NITROGEN

Nitrogen fixation in nature. Classification of nitrogen-fixing bacteria. Iso-
lation of anaerobic bacteria. Morphology of the anaerobic bacteria.
Distribution of anaerobic nitrogen-fixing bacteria in the soil. Physiology
of anaerobic nitrogen-fixing bacteria. Non-symbiotic nitrogen fixing
aerobic bacteria. Description of species of Azotobacter. Morphology
and life cycle of Azotobacter. Physiology of Azotobacter. Other non-
symbiotic nitrogen-fixing bacteria. Symbiotic nitrogen fixation by nodule
bacteria. Historical. Nomenclature. Media. Nodule formation. Iso-
lation of organism from nodules. Isolation from soil. Colony appearance.
Morphology and life cycle of organism. Motility. Physiology of nodule
bacteria. Specific differentiation. Nodule formation by non-leguminous
plants. Nodule formation in the leaves of some plants 103

Chapter V

HETEROTROPHIC, AEROBIC BACTERIA REQUIRING COMBINED NITROGEN

General classification. Spore-forming bacteria. Classification of spore-
forming bacteria. Occurrence of spore-forming bacteria in the soil. Non-
spore-forming bacteria. Classification. Occurrence of non-spore-forming
bacteria in the soil. Thermophilic bacteria. Mycobacteria. Myxobacteria 141

Chapter VI

ANAEROBIC BACTERIA

Oxygen tension in the growth of bacteria. Methods of isolation of anaerobic
bacteria from the soil. Cultivation of anaerobes. Classification of soil
anaerobes. Physiological activities of anaerobic bacteria. Soil processes
in which anaerobic bacteria take an active part 160

Chapter VII

BACTERIA REDUCING NITRATES AND SULFATES

General classification of nitrate reducing bacteria. Organisms reducing
nitrates to nitrites. Organisms reducing nitrates to ammonia. Bacteria
reducing nitrates to atmospheric nitrogen. Description of some typical
denitrifying bacteria. Bacteria reducing sulfates to hydrogen sulfide. . . . 180

Chapter VIII

BACTERIA CAPABLE OF DECOMPOSING CELLULOSES AND OTHER COMPLEX
CARBOHYDRATES AND HYDROCARBONS IN THE SOIL

Microorganisms concerned in the decomposition of celluloses in nature.
Anaerobic bacteria. Aerobic bacteria. Decomposition of cellulose by

CONTENTS XX1U

denitrifying bacteria. Thermophilic bacteria. Pectin decomposing bac-
teria. Bacteria decomposing hydrocarbons and benzene ring compounds. . 190

Chapter IX

BACTERIA DECOMPOSING UREA, URIC, AND HIPPURIC ACIDS

Organisms decomposing urea. Methods of isolation. Occurrence of urea
bacteria. Classification and description. Bacteria decomposing calcium
cyanamide. Uric and hippuric acid bacteria 206

Chapter X

SOIL ALGAE

Introductory. Methods of isolation of impure cultures of algae. Isolation
of pure cultures. Cultivation of soil algae. Distribution of algae in the
soil. Occurrence of algae in the soil. Biochemical activities of algae.
Role of algae in the soil 215

Chapter XI

SOIL FUNGI

Occurrence of fungi in the soil. Methods of demonstrating the occurrence
and abundance of fungi in the soil. Methods of cultivation of soil fungi.
Isolation of single spore cultures. Classification of fungi with special refer-
ence to those occurring in the soil. Occurrence of specific fungi in the soil.
Activities of fungi in the soil. Influence of reaction upon the growth of
fungi. Cellulose decomposition by fungi. Decomposition of nitrogenous
substances (ammonia formation). Utilization of nitrogen compounds by
fungi. Nitrogen fixation. Mycorrhiza Fungi. Nature of mycorrhiza
formation. Plants forming mycorrhiza. Organisms responsible for
mycorrhiza formation. Role of mycorrhiza in the nutrition of plants. . . . 236

Chapter XII

SOIL ACTINOMYCES

General considerations. General description of the genus Actinomyces.
Terminology and systematic position. Species differentiation. Methods
of study. Nature of growth on artificial media. Vegetative mycelium.
Spore bearing mycelium. Utilization of carbon compounds by actinomyces
as sources of energy. Nitrogen utilization. Oxygen requirement. Influ-
ence of temperature, drying and radiation. Influence of reaction and salt
concentration. Influence of poisons. Reduction of nitrates and other
compounds. Production of odor. Pigment formation. Variability.
Species differentiation. Key to the identification of species of soil
actinomyces. Importance of actinomyces in the soil , , , 285

XXIV CONTENTS

Chapter XIII

SOIL PROTOZOA

General morphology of protozoa. Physiology of protozoa. Media for the
cultivation of protozoa. Isolation of pure cultures of protozoa. Staining
of protozoa. Life history of protozoa. Occurrence of trophic and encysted
protozoa in the soil. Classification and occurrence of protozoa in the soil.
Importance of protozoa in the soil 311

Chapter XIV

THE NON-PROTOZOAN FAUNA IN THE SOIL

Animal ecology as a whole and classification of soil forms. Methods of
study. Flatworms. Nematoda. Rotatoria. Annelida. Tartigrada.
Arthropoda. Arachnida. Myriapoda. Insecta. Mollusca. Influence of
environmental conditions on the invertebrate fauna of the soil. Economic
importance of the invertebrate fauna of the soil 341

PART C. CHEMICAL ACTIVITIES OF MICROORGANISMS

Chapter XV

GENERAL PRINCIPLES OF MICROBIAL METABOLISM

Metabolism as a whole. Chemical reactions in the microbial cell. Enzymes
of microorganisms. Reaction velocity. Growth, life and death of micro-
organisms. Chemical composition of the microbial cell 367

Chapter XVI

ENERGY TRANSFORMATIONS IN THE METABOLISM OF MICROORGANISMS

Life and energy. Energy transformations by autotrophic bacteria. Energy
utilization from the oxidation of nitrogen compounds. Energy utilization
from the oxidation of sulfur and its compounds. Energy utilization from
the oxidation of iron compounds. Energy utilization from the oxidation of
hydrogen. Energy utilization from the oxidation of simple carbon com-
pounds. Heterotrophic utilization of energy by microorganisms. Aerobic
utilization of energy. Anaerobic utilization of energy. Efficiency of
energy utilization by heterotrophic microorganisms. Reduction of nitrates
and sulfates and energy utilization. Comparative amounts of energy
liberated by microbiological processes. Energy transformation in syn-
thetic processes. Energy transformation in the soil 384

Chapter XVII

CHEMISTRY OF DECOMPOSITION OF NON-NITROGENOUS ORGANIC MATTER BY SOIL

MICROORGANISMS

Composition of vegetable organic matter. Chemistry of celluloses. Mech-
anism of decomposition of cellulose by microorganisms. Decomposition
of cellulose by anaerobic bacteria. Decomposition of cellulose by aerobic

CONTENTS XXV

bacteria. Decomposition of cellulose by thermophilic bacteria. Decom-
position of cellulose by denitrifying bacteria. Cellulose decomposition
by actinomyces. Cellulose decomposition by filamentous fungi. Cellulose
decomposition in manure. Importance of cellulose decomposition in
the soil. Influence of soil conditions upon cellulose decomposition.
Chemistry of hemicelluloses. Decomposition of hemicelluloses by micro-
organisms. Lignins, ligno-celluloses and their decomposition. Pectins,
mucilages and gums and their decomposition by microorganisms. Starches
and their decomposition by microorganisms. Decomposition of fats and
waxes. Decomposition of paraffins, aliphatic hydrocarbons and benzene
ring compounds in the soil. Decomposition of glucosides and monosac-
charides. Decomposition of organic acids 427

Chapter XVIII

DECOMPOSITION OF PROTEINS AND OTHER ORGANIC NITROGENOUS COMPOUNDS BY

SOIL MICROORGANISMS

Physical and chemical properties of proteins. Chemistry of protein hydrol-
ysis. Protein decomposition by microorganisms. Chemistry of ammonia
formation in the decomposition of proteins by microorganisms. Decom-
position of organic nitrogenous compounds of a non-protein nature. Am-
monia formation by bacteria. Ammonia formation by fungi and actinomy-
ces. Rate of ammonia formation by microorganisms and methods of
determinination. Nitrogen transformation in the rotting of manure.
Nitrogen transformation in the decomposition of organic matter in the
soil. Influence of nitrogenous decomposition products on the growth of
plants and microorganisms 470

Chapter XIX

INFLUENCE OF AVAILABLE ENERGY UPON THE TRANSFORMATION OF NITROGENOUS
COMPOUNDS BY MICROORGANISMS

Carbon and nitrogen transformation by microorganisms. Influence of non-
nitrogenous organic compounds upon the decomposition of nitrogenous
compounds and upon the amounts of ammonia liberated. Decomposition
of organic substances of varying carbon-nitrogen ratio. Different groups
of microorganisms as affecting the carbon nitrogen ratio in the medium.
Influence of straw and plant residues upon the growth of cultivated plants . . 504

Chapter XX

OXIDATION PROCESSES — NITRATE FORMATION

Oxidation-reduction phenomena. Oxidation processes in the soil. Source of
nitrates in the soil. Mechanism of ammonia oxidation. Mechanism of
nitrite oxidation. Nitrate formation from inorganic salts and from organic
nitrogenous compounds. Influence of reaction on nitrate formation.
Influence of organic matter upon nitrate formation. Influence of salts.
Influence of soil gases. Nitrate formation in solution and in soil. Influ-
ence of soil treatment upon nitrification in the soil. Oxidation of sulfur
and other minerals in the soil. Oxidation of organic compounds in the soil. 520

XXVI CONTENTS

Chapter XXI

REDUCTION PROCESSES — NITRATE REDUCTION

Reduction processes in the soil. Transformation of nitrates by micro-
organisms. Nitrate assimilation. Utilization of nitrates by micro-
organisms as sources of oxygen. Reduction of nitrates to gaseous nitro-
gen and oxides of nitrogen. Formation of nitrogen gas from organic com-
pounds. Denitrification in the soil. Importance of nitrate-reduction in
the soil. Reduction of other substances in the soil 542

Chapter XXII

FIXATION OF ATMOSPHERIC NITROGEN BY MICROORGANISMS

Non-symbiotic fixation of nitrogen. Source of energy. Chemistry of de-
composition of carbohydrates. Respiration and nitrogen fixation. Pro-
tein synthesis by Asiotobacter. Chemistry of process of non-symbiotic
nitrogen fixation. Influence of available nitrogen compounds upon nitro-
gen fixation. Influence of salts upon nitrogen fixation. Influence of
organic matter upon nitrogen fixation. Influence of reaction. Influence
of moisture and temperature upon nitrogen fixation. Soil cultivation and
nitrogen fixation. Importance of non-symbiotic nitrogen-fixation proc-
esses in the soil. Symbiotic Nitrogen Fixation. Relation between the
bacteria and the host plant. Chemistry of nitrogen-fixation by symbiotic
bacteria. Production of gum by legume bacteria. Influence of reaction
on the growth of Bad. radicicola and nodule formation. Nodule formation
and nitrogen fixation. Influence of environmental conditions. Importance
of symbiotic nitrogen fixation in the soil. Associative action of legumes
and non-legumes 558

Chapter XXIII

TRANSFORMATION OF SULFUR BY MICROORGANISMS

Sources of sulfur in the soil and processes of transformation. The nature of
oxidation of sulfur and its compounds in the soil. Reduction of sulfur and
its compounds. Formation of H2S in the decomposition of organic matter.
Sulfur oxidation and transformation of minerals 600

PART D. SOIL MICROBIOLOGICAL PROCESSES AND SOIL FERTILITY

Chapter XXIV

THE SOIL AS A MEDIUM FOR THE GROWTH AND ACTIVITIES OF MICROORGANISMS

The soil as a culture medium. Soil composition and microbiological activi-
ties. The mineral composition of the soil. The physico-chemical role of
organic matter in the soil. Colloidal condition of soils and microbiological
" activities. Soil solution. Soil reaction and microbiological activities.
The soil atmosphere. Soil temperature. Growth of microorganisms in soil
in pure and mixed culture. The idea of a soil population 619

CONTENTS XXV11

Chapter XXV

TRANSFORMATION OF MINERALS IN THE SOIL

Nature of mineral transformation by microorganisms. Decomposition of
rocks and rock constituents by microorganisms. Nature of phosphorus
compounds in the soil. Decomposition of organic phosphorus compounds
by microorganisms. Transformation of insoluble tri-calcium phosphates
into soluble forms by microorganisms. Transformation of insoluble phos-
phates by inorganic and organic acids formed by microorganisms. Trans-
formation of potassium in the soil by microorganisms. Transformation of
calcium in the soil. Transformation of magnesium in the soil. Transfor-
mation of manganese in the soil. Transformation of zinc. Transformation
of iron. Transformation of aluminum in the soil. Role of minerals in
bacterial metabolism 644

Chapter XXVI

TRANSFORMATION OF ORGANIC MATTER IN THE SOIL

Nature of soil organic matter. Decomposition of organic matter added
to the soil. Transformation of the various constituents of the organic
matter added to the soil. Evolution of carbon dioxide as an index of
decomposition of organic matter in the soil. Formation of ammonia
(and nitrate) as an index of decomposition of organic matter in the soil.
Formation of "humus" as an index of decomposition of organic matter
in the soil. Nature of soil "humus." Chemistry and classification of
humus compounds. Soil organic matter and the activities of microorgan-
isms. Carbon-nitrogen ratio in the soil 669

Chapter XXVII

MICROBIOLOGICAL ANALYSIS OF SOIL AS AN INDEX OF SOIL FERTILITY

Soil fertility and microbiological activities. Methods for determining the
microbiological condition of the soil. Numbers of microorganisms in the
soil. Nitrifying capacity of the soil. Evolution of carbon dioxide.
Cellulose decomposing capacity of the soil. Nitrogen fixing and mannite
decomposing capacity of the soil. The catalytic action of the soil. Oxi-
dizing and reducing power of the soil 708

Chapter XXVIII

SOIL MICROBIOLOGICAL EQUILIBRIUM; INFLUENCE OF AIR DRYING AND PARTIAL
STERILIZATION UPON THE ACTIVITIES OF MICROORGANISMS IN THE SOIL

Microbiological equilibrium in the soil. Influence of air drying of soil upon
the microbiological equilibrium. Influence of caustic lime upon soil
processes. Partial sterilization of soil. The use of heat as an agent of
partial sterilization. Influence of volatile antiseptics upon bacterial ac-
tivities in the soil. Protozoan theory. Agricere and bacteriotoxin theory.
Destruction of selective groups of organisms. Interrelationships of micro-
organisms in the soil 738

XXV111 CONTENTS

Chapter XXIX

INFLUENCE OF ENVIRONMENTAL CONDITIONS, SOIL TREATMENT, AND PLANT GROWTH
UPON MICROORGANISMS AND THEIR ACTIVITIES IN THE SOIL

Influence of organic matter upon the soil population. Influence of stable
manure. Influence of temperature. Influence of moisture. Influence of
soil cultivation. Influence of salt concentration upon the activities of
microorganisms in the soil. Influence of calcium oxide and carbonates of
calcium and magnesium. Influence of growing plants upon soil micro-
organisms and their activities 767

Chapter XXX

SOIL AS A HABITAT FOR MICROORGANISMS CAUSING PLANT AND ANIMAL DISEASES

Influence of saprophytic soil microorganisms upon plant growth. Sapro-
phytism and parasitism among soil microorganisms. Animal and plant
diseases caused by bacteria that may be found in the soil. Plant diseases
caused by fungi found in the soil. Plant and animal diseases caused by
species of actinomyces. Plant and animal diseases caused by invertebrate
animals found in the soil. Relation of soil environment to plant infection.
Influence of reaction upon the growth of plant pathogenic organisms in the
soil. Methods of control SOI

Chapter XXXI

SOIL INOCULATION

Beneficial and injurious microbiological processes in the soil. Introduction
of certain useful microorganisms into the soil. Legume inoculation. Use
of soil for inoculation of legumes. Commercial cultures and their prepara-
tion. Biological types of legume bacteria. Importance of legume inocula-
tion. Inoculation of non-leguminous plants with nodule bacteria. Inocu-
lation of soil with non-symbiotic nitrogen-fixing bacteria. Soil inoculation
with autotrophic bacteria. Inoculation of soil with heterotrophic, non-
nitrogen-fixing microorganisms 817

Chapter XXXII

HISTORY OF SOIL MICROBIOLOGY, ITS PAST, PRESENT AND FUTURE

Beginnings of soil microbiology. Soil microbiology as an independent
science. Recent advances of the science. Present outstanding problems
in soil microbiology 834

PART A

OCCURRENCE AND DIFFERENTIATION

OF MICROORGANISMS

IN THE SOIL

". . . le role des infiniments petits m'apparaissait infiniment grand . .

— Pasteur.

CHAPTER I

Numbers of Different Groups of Microorganisms Found in the
Soil and Methods of Determination

The occurrence of microorganisms in the soil. The microorganisms
present in the soil belong, in an uneven proportion, to the plant and
animal kingdoms, the former including the large majority both in
numbers and in kinds. Chart 1 gives a visual representation of the
relationships of the various groups of soil microorganisms. The
relative importance in the soil, however, both as to numbers and
physiological activities, varies with the different groups.

The animal world is represented in the soil by the protozoa, nema-
todes, rotifers, earthworms and various other worms as well as insects.
The nematodes occur abundantly in all soils, but especially in green-
house soils and certain infested field soils. Large numbers as well as
numerous species of amoebae, ciliates and flagellates represent the
protozoa in the soil.

The microscopic plant world is represented in the soil by the algae,
fungi and bacteria, named in the order of their increasing importance
of numbers and activities. Among the algae, the Cyanophyceae and
Chlorophyceae are best represented in the soil. The soil fungi can be
subdivided further into three groups :

1. Yeasts and yeast-like fungi, like the Monilia and Oidia (these
two groups may, however, be classed with the true fungi).

2. Molds and other true fungi. Here we find the Mucorineae repre-
sented by the extensive genera Rhizopus, Mucor, Zygorhynchus and
other Phycomycetes; various Ascomycetes, including the genus Chae-
tomium and other genera; Hyphomycetes represented by the Mucedi-
naceae (Aspergillus, Penicillium, Sporotrichum, Botrytis, Trichoderma,
Verticillium, etc.), Dematiaceae, Stilbaceae and Tuberculareaceae.
The Basidiomycetes are probably represented abundantly in the soil
by the sterile mycelium as well as by some of the mycorrhiza fungi.

3. Actinomyces. Ten to 50 per cent of the colonies developing from a
soil on the common agar or gelatin plate belong to this important group
of soil organisms. They are generally classified by bacteriologists with

3

4 PRINCIPLES OF SOIL MICROBIOLOGY

the bacteria; actually they belong to the fungi and are so far known
to be represented in the soil by one extensive genus Actinomyces.

Bacteria predominate, in numbers and in the variety of activities,
over all the other groups of microorganisms. This was the reason why
the earlier microbiologists named the whole science of soil microbiology
"soil bacteriology." It has long been recognized, however, that the
soil population consists of various microorganisms other than bacteria,
so that the more comprehensive term is fast coming into general use.
Since the bacterial activities in the soil do not coincide with their
taxonomic groupings, these organisms may be classified on the basis
of their physiology for the sake of convenience in treatment. As a
major division, the bacteria can be separated into two large groups:

Chart I. The microflora and microfauna of the soil

(1) autotrophic, and (2) heterotrophic forms. Living organisms that
require for their nutrition substances which have been built up by other
organisms are called heterotrophic. The heterotrophic saprophytic
bacteria consume, for their energy and for the building up of their
protoplasm, the organic compounds of plant and animal bodies. Organ-
isms like the green plants and certain bacteria that can thrive on purely
inorganic substances and obtain their carbon from the carbon dioxide
of the atmosphere are called autotrophic. But while the green plants
derive their energy photosynthetically, the autotrophic bacteria derive
their energy from the oxidation of purely inorganic substances, or
chemosynthetically. The autotrophic group of bacteria is represented
in the soil by smaller numbers and by much fewer species than the
heterotrophic group, but it includes forms which are of greatest impor-

NUMBERS OF MICROORGANISMS 5

tance in the physiological processes in the soil, namely the organisms
which oxidize ammonium salts to nitrites, nitrites to nitrates, sulfur
and sulfur compounds to sulfates, and a few other less important groups.

The heterotrophic bacteria are further subdivided on the basis of
their nitrogen utilization: (1) Those bacteria that are able to fix
atmospheric nitrogen in the presence of sufficient carbohydrates as
sources of energy. This division is again only secondary in numbers,
but its three representative groups play an important part in the soil
economy, namely in the increase of the combined nitrogen of the soil.
They are the symbiotic nitrogen-fixing, or nodule bacteria; the non-
symbiotic aerobic nitrogen-fixing bacteria and the non-symbiotic
anaerobic nitrogen-fixing bacteria. (2) Those bacteria which depend,
for their metabolism, upon the nitrogen of the soil, in organic or inor-
ganic forms. The heterotrophic non-nitrogen-fixing bacteria can be
further subdivided, using as a basis either the need of free or com-
bined oxygen or spore formation. The heterotrophic, non-nitrogen-
fixing, aerobic bacteria are usually the organisms which are found on
the plates, when an analysis of numbers of bacteria in the soil is made
by the common agar or gelatin plate method.

In addition to the microscopic forms, ultramicroscopic microorganisms
capable of passing through bacterial filters have been reported 1 as
present in the soil. These have been only insufficiently studied. We
may be dealing here with certain stages of other organisms, as sug-
gested by Lohnis for gonidia. A certain relation was observed between
the ultrafilterable microbes and microbial enzymes and other cell
constituents. Attention may be called here to the extensive literature
concerning the nature of the bacteriophage; investigators do not agree
as yet whether these are ultramicroscopic organisms or are of the nature
of enzymes. An attempt to study the physiological activities of the
invisible soil microorganisms has been made 2 but without any success.

Proof of microbial activities in the soil. The food requirements of the
various groups of soil microorganisms are so distinctly different that
no single artificial culture medium could be devised on which all of them
could be studied. A large number of microorganisms, to which some
of the most important soil forms belong, will grow only under very
special conditions, such as selective media or selective environments.

1 Melin, E. Ultramikroskopische Mikroben im Waldboden. Ber. deut. Bot.
Gesell. 40: 21-25. 1922. See also Miehe, H. Biol. Centrbl. 43: 1-15. 1923.

2 Rossi, G. Preliminary note on the microbiology of the soil and the possible
existence therein of invisible germs. Soil Sci. 12: 409-412. 1921.

6 PRINCIPLES OF SOIL MICROBIOLOGY

Various media and different methods have to be used for the study of the
different groups. In some cases, special enrichment culture media favor-
ing the development of particular organisms have to be devised, so that
the growth of these will take place in preference to that of all the
other organisms. We thus often create artificial conditions which are
distinctly different from those of the soil and conclusions, based on the
results of growth of the organisms under such artificial conditions, often
do not hold true for the soil. To be able to grow the organisms in pure
culture in the soil, the latter must be first sterilized. No method of
sterilization has yet been devised which would not modify, in a funda-
mental manner, the chemical conditions of the soil. What will hold
true for sterilized soil, then, may not hold true for unmodified soil.
Again, the various organisms exist in the soil in large numbers, with a
number of associative and antagonistic influences at work (both by
living microorganisms and their products). Each organism has
adapted itself to its environmental conditions and to the other organisms
and may be, so to speak, in a condition of "unstable equilibrium."
When this same organism is cultivated, in pure culture, upon a favor-
able medium, its activities are very likely to be different from those in
the normal soil. Before we can conclude that a microorganism is
active in the soil and that certain chemical transformations are produced
by this organism under ordinary soil conditions, certain requirements
must be satisfied. The following postulates, applied by Koch to
pathogenic bacteria, and modified by Conn 3 in their application to soils
should hold true for soil microorganisms: (1) The organism must be
shown to be present in the soil in an active form when the chemical
transformation under investigation is taking place. (2) The organism
must be shown to be present in larger numbers in such soil than in
similar soil in which the chemical change is not taking place. (3)
The organism must be isolated from the soil and studied in pure cul-
ture. (4) The same chemical change must be produced by the or-
ganism in experimentally inoculated soil, making the test, if possible,
in unsterilized soil. (5) The organism must be found in the inoculated
soil.

Methods of study. The methods generally employed for the study of
soil bacteria can be divided into those of direct microscopic observation
and cultural methods. The former have been suggested by Conn and
further developed by Winogradsky. The latter have been used by the

3 Conn, H. J. The proof of microbial agency in the chemical transformation
of soil. Science. N. S. 46: 252-255. 1917.

NUMBERS OF MICROORGANISMS 7

great majority of other soil microbiologists. Artificial culture media are
employed, or at least artificial conditions are created. In many in-
stances, therefore, no direct evidence is furnished as to what is actually
taking place in the soil, under natural conditions. The results obtained
under laboratory conditions often have to be interpreted as to their
bearing upon actual field results.

The information obtained from the study of soil microbiology by the
use of the different methods can throw light upon three groups of
phenomena: (1) the numbers and kinds of microorganisms occurring
in the soil; (2) the activities of soil microorganisms; (3) the bearing of
these activities upon soil fertility.

Direct microscopic method.

The method consists in preparing; a suspension of soil in a dilute fixative solu-
tion, then spreading one or two drops of the suspension upon a clean slide, drying
and staining with an acid dye. For qualitative purposes, about 0.5 to 1 gram of
soil is placed 4 in a test tube; 6 to 8 cc. of a fixing solution, consisting of 0.04 per
cent sterile gelatin in water, are then added and the mixture well shaken. Two
loopfuls of the suspension are placed upon clean slides; after drying, the slides
are stained with a 1 per cent solution of rose bengal in 5 per cent phenol-water
mixture. The preparation is heated on a steam bath until most of the liquid has
evaporated and the excess of stain is removed by dipping the slide in water.
The preparation is then dried on the steam bath and examined microscopically.
The gelatin fixative can be omitted 5 and the films fixed to the slide by flooding,
after drying, with a very dilute solution of collodion in ether and alcohol.

The method was modified and improved by Winogradsky, 6 who found that the
presence of large yellow grains of inorganic soil material hinders the proper
examination of the field under the microscope. The soil samples are well mixed
and powdered. One gram of the soil (on a dry basis) is then added to 4 cc. of
distilled water and shaken vigorously for five minutes. After allowing to rest
30 seconds the suspension covering the large sedimented inorganic particles is
poured off into a small tube of a hand centrifuge. Two 3-cc. portions of distilled
water are then added to the residue, shaking each time one minute, allowing to
rest 30 seconds and then pouring into the same tube of the centrifuge. Ten
units of water are thus used for one unit of soil. After these three washings the

4 Conn, H. J. The microscopic study of bacteria and fungi in soil. N. Y.
Agr. Exp. Sta. Tech. Bui. 64. 1918; An improved stain for bacteria in soil.
Stain Technol. 1: 126-128. 1926.

5 Whittles, C. L. The determination of the number of bacteria in soil. Jour.
Agr. Sci. 13: 18-48. 1923; 14: 346 369. 1924.

8 Winogradsky, S. Sur l'etude microscopique du sol. Compt. Rend. Acad.
Sci. 179: 367-371. 1924; Etudes sur la microbiologic du sol. 1. Sur la methode.
Ann. Inst. Past. 39: 299-354. 1925.

8 PRINCIPLES OF SOIL MICROBIOLOGY

first sediment suspended in distilled water settles immediately. During these
manipulations, which require about 10 minutes, a second sediment is formed
in the tube of the centrifuge. About half of the suspension is carefully taken
out and placed in another centrifuge tube; on centrifuging, a third sediment is
formed. Preparations are then made from each sediment and from the non-
centrifuged and centrifuged suspensions. One drop of the various preparations
is placed upon a slide covering just 1 sq. cm. ; the preparations are dried in an oven
and are rapidly covered with a very dilute agar solution. One per cent warm
agar solution is best for the first two sediments and 0.1 per cent cold agar solution
for the third sediment. For the suspensions, no fixative is necessary. When the
agar is dried, several drops of absolute alcohol are used for fixing and the prepa-
ration is stained by means of a solution of an acid dye in 5 per cent phenol solution.
Rose bengal may be used, but its action is prolonged, followed by a drop of acetic
acid, then washed. Extra erythrosine in 5 per cent phenol solution is superior.
The bacterial cells are colored, but not the capsules and mucus; this is especially
true of the compact colonies as those of Nitrosomonads and other soil forms
which so readily over-color with basic dyes; the colloids are only faintly colored;
the agar is readily discolored by the process of washing with cold water. The
dye is allowed to act 5 to 15 minutes in the cold or on slight warming, then washed
a few seconds in water.

The preparations from the first sediment are usually free from bacteria, except
in soils rich in organic matter, when some of the particles are not removed by
three washings. The second preparation shows on examination the same mi-
crobes, qualitatively and quantitatively, as the third sediment, where conditions
for examination are most favorable. The fourth preparation made from the
suspension is usually most instructive. The living cells only take the stain,
while the spores stain only very faintly or not at all and can be seen only when
present in large numbers. Protozoan cysts are recognized by their intense
coloration and can easily be counted.

Winogradsky suggested to use always for comparison a control soil,
which had no addition of fresh organic matter for a considerable period
of time. A normal arable soil contains a native or autochtonous flora
consisting of short bacteria with rounded ends and of cocci, 1 to 1.5/z in
diameter. Often larger forms, 1 to 3/x in diameter, resembling Azoto-
bacter are found. They group into rounded colonies consisting of
about 100 cells in a compact mass with a common capsule, but occa-
sionally with as few as a dozen individuals (PI. I). The field between is
completely devoid of microbes. The colonies are situated on the soil
colloidal matter. This is the reason why the centrifuged suspension is
practically free from colonies which are carried down by the flakes of
organic matter. Spore-bearing bacilli, filamentous bacteria, spirals,
mycelial filaments, actinomyces, and protozoan cysts are absent
or are very rare. The presence of these indicates that the soil is in an
active state of fermentation, due to recent addition of organic matter.

PLATE I

• • ;

V #

*

3

1. The bacteria grow in the soil, in the form of zooglea-like masses, upon the
colloidal material surrounding the inorganic soil particles, as shown by the
direct microscopic method, X 1200 (from Winogradsky) .

2. Zooglea-like mass of bacteria in soil, as shown by the direct microscopic
method, X 1200 (from Winogradsky).

3. Large cells of bacteria in Texas sandy soil (Azotobacter?), as shown by
direct microscopic examination, X 1200 (from Winogradsky).

4. The distribution of organic matter and bacteria in the soil (Russian
tshernoziem) (after Winogradsky).

NUMBERS OF MICROORGANISMS 9

The application of the direct microscopic examination to the study
of occurrence and distribution of microorganisms in the soil gives more
direct evidence as to the presence and relative abundance of specific
groups of microorganisms. The direct microscopic examination has
been used 7 for counting bacteria in animal feces; it can also be used
for counting various microorganisms in culture. To determine the
numbers of microorganisms quantitatively by the use of the direct
microscopic method, various difficulties are encountered:

1. Some of the microorganisms, like the protozoa, will be destroyed in the
process of staining.

2. Others, like the fungi, may prove too large, for the very small quantity of
soil that can be used for the examination.

3. The bacteria themselves are found in clumps upon the colloidal film and not
in the soil solution. Not only is it difficult to count the bacteria in the film, but
the variability is so great that it would take a large number of counts to obtain
reliable results

The same procedure is followed as for qualitative determinations.

The soil is diluted by means of a weak solution of gelatin (0.15 gram gelatin in
1000 cc. of hot water and kept sterile in a cotton plugged flask) using one part of
soil to 3 to 10 parts of solution depending on the soil type, heavier soils requiring
a higher dilution. The smear is prepared from 0.1 cc. of the infusion measured
out from a thin graduated pipette, to cover 1 sq. cm. on a clean slide, previously
rinsed in alcohol; the smears are allowed to dry over a steam bath. For staining
either rose bengal (1 gram in 100 cc. of 5 per cent phenol solution) or erythrosine
can be employed. The stain is allowed to act 1 to 3 minutes, then washed and
dried. With the rose bengal stain the bacteria are found deep pink or red, the
mineral particles uncolored, some of the dead organic matter light pink but most
of it yellow or unstained. The preparations are examined with an oil immersion
objective and a high-power eye-piece. By means of a simple equation, the
number of organisms can then be determined. It is advisable not to count the
entire field, but to mark off the central portion. 8 A disc with circles and cross
lines is placed in the eye-piece. Conn suggested to use a circle of such a size as to
cover an area on the slide either 80 or 113 microns in diameter. Every organism
in the area will represent two and one millions respectively per cubic centimeter,
using a 1.9 ( T V inch) fluorite objective CN.A. 1.32) with a 12.5 X ocular. This
quantity is multiplied by the dilution of the soil to give the number of bacteria
per gram of soil.

However, the uneven distribution of the bacteria in the soil, causing
great irregularities and the difficulty of distinguishing bacterial cells from

7 Klein, A. Die physiologische Bakteriologie des Darm-Kanals. Arch. Hyg.
45: 117. 1902.

8 Breed, R. S., and Brew, J. D. Counting bacteria by means of the microscope.
N. Y. Agr. Exp. Sta., Tech. Bui. 49. 1916.

10 PRINCIPLES OF SOIL MICROBIOLOGY

soil particles, especially in case of clay soils, and of separating living from
dead bacteria make accurate counts impossible. The method can,
therefore, not be used as yet for quantitative work, but is quite applica-
ble for qualitative purposes, to show the types of microorganisms which
exist in the soil in an active form. The microscopic method may be
used for counting bacteria in culture media, especially in a liquid form,
but even here the total volume of microbial cells may prove 9 a better
index of the activities of the organisms than their numbers.

Organisms found in the soil by the direct microscopic method. Conn
demonstrated that the actual number of bacteria found in the soil, by
the use of the microscope, is probably five to twenty times as great
as that indicated by the culture plate method. This discrepancy is
due to that fact that a large number of soil bacteria do not grow on the
plates. By far the greatest number of microorganisms found in the
soil, by the use of the microscope, consists of the minute non-spore-
forming rods and cocci. The large spore-forming bacteria (as Bac.
megatherium and Bac. cereus) have been found in normal soil only in the
form of spores, which make up a very small proportion of the total
bacterial flora of the soil. Filaments of actinomyces have also been
found, but to a lesser extent than the spores of these organisms. Fungus
mycelium was not found in any soil, except when an unusual amount of
organic matter is present. The spore-forming bacteria become 10 active
in the soil only when a great excess of easily decomposable organic
matter has been added or when the moisture content of the soil is high.
The minute non-spore-forming rods and cocci are considered 11 to form
the autochtonous microflora of the soil.

Other investigators found that the microscopic examination of soil
bacteria allows the differentiation of three distinct groups, 12 namely (a)
cocci and short rods, (b) typical large cells of Azotobacter and (c)
bacillary forms. The first two groups are largely connected with the

9 Skar, O. Mikroskopische Zahlung und Bestimmung des Gesamtkubikin-
haltes der Mikroorganismen in festen und fliissigen Substanzen. Centrbl. Bakt.,
II, 57 : 327-344. 1922. Fries, K. A. Eine einfache Methode zur genauen Bestim-
mung der Bakterienmengen in Bakteriensuspensionen. Centrlbl. Bakt. I (Orig.),
86: 90-96. 1921.

10 Winogradsky, S. Sur la microflore autochtone de la terre arable. Compt.
Rend. Acad. Sci. 178: 1236-39. 1924.

11 Joffe, J. S., and Conn, H. J. Factors influencing the activity of spore form-
ing bacteria in soil. N. Y. Agr. Exp. Sta. Bui. 97. 1923.

12 Richter, A. A. and B. A. To the question of microscopic soil investigation.
(Russian). Utchonie Zapiski, Saratov Univ. 4: No. 1. 1925.

NUMBERS OF MICROORGANISMS

11

colloidal soil particles, in the form of zooglea, surrounded by slimy
capsules; the third group is found mostly in the soil solution, but the
representatives of this group occur frequently also in the form of
clumps, especially on decomposing organic matter. A comparative
study of the occurrence of these three groups at different depths of soil
has given the following results :

TYPE OF SOIL

Forest soil.

Brown loam soil.

Sandy soil.

Surface
10 cm.
20 cm.

Surface
10 cm.

Surface
10 cm.
20 cm.

NUMBERS OF BACTERIA. IN
MILLIONS PER GRAM

YEAST
CELLS

Cocci

Azoto-
bacter
cells

Bacilli

1,379
991
281

870
569

519
407
269

156

82
188

188
184

155

112

51

1,212
466
169

376
106

192
153
139

millions
per gram

1

31

84
1

79
23

8

PIECES OF

FUNGUS
MYCELIUM

millions
per gram

47
34

7

3

19
3

The non-spore forming bacteria are thus found to be most abundant,
the large rods or bacillary forms coming next, especially in soils rich in
decomposing organic matter. Fungus mycelium is also abundant in
such a soil.

Cultural methods for demonstrating the kinds of organisms active in the
soil. The cultural methods for the study of soil microorganisms are
divided into methods for (a) quantitative study of soil microorganisms,
(6) qualitative studies, and (c) for the study of microbiological activities,
both in pure culture and in the soil.

Winogradsky 13 suggested to use the silica gel plate, to which a specific
substance is added, for demonstrating the existence of specific organisms
in the soil.

Pure, colorless potassium silicate is dissolved in water to a specific gravity of
1.06 (6 to 8 Beaume). A dilution of HC1 equivalent to a specific gravity of 1.10
(13 Beaume) is also prepared. An equal volume of the silicate is poured into the

13 Winogradsky, S. Sur une methode pour apprccier le pouvoir fixateur de
l'azote dans les terres. Compt. Rend. Acad. Sci. 180: 711-716. 1925.

12 PRINCIPLES OF SOIL MICROBIOLOGY

acid solution and both are well mixed. The mixture is then distributed into
Petri dishes and these are allowed to rest over night. A firm gel is obtained.
The uncovered dishes containing the gel are then placed in flowing water for at
least twenty-four hours, until no reaction is given with methyl red or brom cresol
purple and with AgN0 3 . A solution containing the minerals and specific sub-
stance, either in solution or as an insoluble suspension, may then be poured
over the surface of the gel and the dishes placed in an oven at 60° to 65°C, until
the excess moisture has dried off. The gel in the dish is inoculated with small
particles of soil, the dish is covered and placed in an incubator. After a few days,
the specific organism, if present in the soil, will develop on the gel surrounding
the particles of soil. By this method the presence of Azotobacter in soil can be
readily demonstrated, provided mannite and CaC0 3 are employed in addition to
the minerals. Nitrite-forming bacteria will develop in an ammonium salt
medium, nitrate-forming organisms in a nitrite medium, etc. 14

However, the cultural methods, largely the enrichment media de-
veloped by Winogradsky and Beijerinck, and the common gelatin and
agar plate have been used most extensively for establishing the presence
and abundance of specific organisms in the soil.

Cultural methods for the determination of numbers of microorganisms
in the soil. The earliest investigations in soil bacteriology were car-
ried out, 15 - 16 . 17 by the use of methods developed in medical bacteriology.
Soils were diluted with sterile soil, then plated out with gelatin and
numbers determined after a certain incubation period. Later, sterile
water was used for making the dilutions. In some cases small quantities
of soil were weighed directly for the preparation of the plates. The
method itself was imperfect and the results unrepresentative, and no
relation was established between numbers and soil productivity.
Hiltner and Stormer 18 suggested the use of the dilution method, with the
hope of doing away with the plate method, but here again the hetero-
trophic bacteria were determined by their growth on agar or gelatin

14 The gel may also be prepared by methods described elsewhere (p. 196).
16 Koch, R. Zur Untersuchung von pathogenen Organismen: Bodenunter-
suchung. Mitt. K. Gesundheitsamt. 1: 34-36. 1881.

16 Proskauer, B. Uber die hygienische und bautechnische Untersuchung des
Bodens auf dem Grundstucke der Charite und des sogen. "Alten Charitd-
Kirchhofes." Bakteriologisches Verhalten des Bodens. Ztschr.Hyg.il: 22-24.
1882.

17 Frankel, C. Untersuchungen iiber das Vorkommen von Mikroorganismen
in verschiedenen Bodenschichten. Ztschr. Hyg. 2: 521-582. 1887.

18 Hiltner, L., and Stormer, K. Studien fiber die Bakterienflora des Acker-
bodens,mit besonderer Beriicksichtigung ihres Verhaltens nach einer Behandlung
mit Schwefelkohlenstoff und nach Brache. Arb. Biol. Abt. Land. r. Forstw., K.
Gesundheitsamt. 3: 445-545. 1903.

NUMBERS OP MICROORGANISMS 13

media, while the autotrophic and nitrogen-fixing organisms were found
not to be able to develop readily in high dilutions. Each of these two
methods (plate and dilution) for determining the number of microor-
ganisms in the soil has certain advantages and disadvantages.

The plate method consists in diluting the soil with sterile tap water,
making a series of dilutions, so that 1 cc. of the final dilution, when
plated out with nutrient agar or gelatin, will allow 40 to 200 colonies
to develop on the plate. The dilution method consists of diluting the
soil first with sterile water, as with the plate method, but transferring
1 cc. of several of the final dilutions into special sterile nutrient media
adapted for the growth of particular groups of microorganisms. The
number of microorganisms will be found to lie between the two highest
dilutions, one of which gives positive and the other negative growth.
This allows us to determine approximately the number of organisms
belonging to each group and present in the particular soil. 19 The latter
method is rather cumbersome, since it involves the preparation of a
large number of media and the use of a number of containers for the
development of various physiological groups of organisms for making
the various dilutions, also, it involves great variability in the results. 20
The plate method is convenient, but its chief limitation is the fact that
it allows the development of only the heterotrophic, non-nitrogen fixing,
aerobic bacteria and of yeasts, molds and actinomyces. The dilution
method can be used for the study of practically all known soil forms.
The two methods may then be used each for its particular purpose,
particularly in view of the fact that, for those microorganisms that
develop on the common culture plate, the dilution method was not
found 18 to give higher results than the plate method. The latter
method should, therefore, be utilized for a general study of the numbers
of microorganisms in the soil, keeping in mind its limitations, while the
dilution method should be used for the determination of the abundance
of special groups of microorganisms which do not develop on the plate.

Culture media. With the introduction by Koch in 1881 of the gelatin
plate for counting bacteria in general, an impetus was also given to the
study of soil bacteria. But, unfortunately, Koch himself and prac-
tically all the bacteriologists following him for the next fifteen years were
medical men interested particularly in the possible presence of patho-

19 Lohnis, F. Zur Methodik der bakteriologischen Bodenuntersuchung.
Centrbl. Bakt. II, 14: 1-9. 1905.

20 Fischer, H. Zur Methodik der Bakterienzahlung. Centrbl. Bakt. II, 25:
457-459. 1910.

14 PRINCIPLES OF SOIL MICROBIOLOGY

genie bacteria in the soil and their importance as carriers of infection.
They quite properly, from their point of view, used the methods of
medical bacteriology. But even the excellent and stimulative researches
of Koch and those following him 21 could not lay a proper foundation of
soil microbiology, due primarily to the lack of proper methods.

The meat-extract-peptone agar or gelatin, found so valuable in patho-
genic bacteriology, is entirely inappropriate for soil work, for various
reasons, chief among which is the fact that the medium is not standard
in composition and that it allows a rapid development of a few organ-
isms which readily overgrow the plate and thus may prevent entirely
the development of others. The distinct inferiority of bouillon agar or
bouillon gelatin can be seen from the results of Engberding, 22 who found
that a soil giving 99 colonies with Heyden agar, gave 39 with bouillon
agar and only two with bouillon gelatin. But even the Heyden agar is
not definite in composition, although it is often used for counting soil
bacteria.

The media used for the determination of numbers of microorganisms
in the soil (those that develop on the plate) should allow the development
of the greatest possible number of organisms and should be standard in
composition, so that every batch made up in the same laboratory or
at any other laboratory will be like every other batch. This means that
inorganic salts should be used. If organic substances are necessary,
they should be pure, stable, and standard if possible, as in the case of
the carbon and nitrogen sources. Various sugars or organic acids used
as sources of carbon can be obtained in a standard form. Nitrogen
substances should also be as standard as possible and used in as small
amounts as possible. Agar in itself should not serve as a nutrient and
should be, therefore, as pure as possible. The objection to gelatin
is that it serves also as a nitrogen source for many microorganisms,
thus making the medium not standard. It should, therefore, be used
only in qualitative work or in special instances, as in the study of the
number of gelatin-liquefying organisms in the soil. To hold in check
the development of certain rapidly growing organisms, which prevent
the growth of the numerous but slow growing bacteria in the soil, the
organic matter content of the media had to be reduced to a minimum.

21 Houston, A. C. Chemical and bacteriological examination of soils. Local
Gov't. Board, Rept. 27: 251-296. 1898.

"Engberding, D. Vergleichende Untersuchungen liber die Bakterienzahl im
Ackerboden in ihrer Abhangigkeit von aussern Einfiiissen. Centrbl. Bakt. II,
23: 569 642. 1909.

NUMBERS OF MICROORGANISMS 15

The first important modifications in the composition of the medium
for a quantitative determination of soil bacteria were made by the
introduction of the soil infusion agar, 23 and later by the elimination
even of the soil extract, 24 using a synthetic agar, with only 0.05 gram of
peptone per liter. The soil extract media do not, however, meet the
qualification of being "standard in composition," since the soil infusion
varies with the soil used for making the infusion. The synthetic
medium was further modified 25 by the substitution of egg-albumin and
casein for peptone. Among the other synthetic media suggested for the
quantitative estimation of soil bacteria and actinomyces, sodium
asparaginate agar, 26 asparaginate-mannite agar, 27 and urea nitrate
agar 28 should be mentioned. For the estimation of fungi, special acid
media have to be used. For the protozoa, the dilution method still
remains the most reliable, and nutrient agar or special liquid media
can be employed for the development of the organisms in the final
dilutions.

Composition of synthetic media

I. Fischer's soil extract agar:

Soil extract 1000 cc.

Agar 12 grams

K 2 HPO« 2 grams

The soil extract is prepared by heating soil for half an hour at 15 pounds pres-
sure with an equal weight of a 0.1 per cent solution of Na 2 C0 3 .

23 Fischer, H. Bakteriologisch-chemische Untersuchungen. Bakteriologis-
cher Teil. Landw. Jahrb. 38: 355-364. 1909.

!4 Lipman, J. G., and Brown, P. E. Media for the quantitative estimation
of soil bacteria. Centrbl. Bakt. II, 25: 447-454. 1910.

25 Brown, P. E. Media for the quantitative determination of bacteria in soils.
Centrbl. Bakt. II, 38: 499-506. 1913; also Iowa Agr. Exp. Sta., Res. Bui. 11,
396-407. 1913. Waksman, S. A. Microbiological analysis of soils as an index
of soil fertility. II. Methods of the study of numbers of microorganisms in the
soil. Soil Sci. 14: 283-298. 1922; Waksman, S. A., and Fred, E. B. A tentative
outline of the plate method for determining the number of microorganisms in the
soil. Ibid. 14:27. 1922.

26 Conn, H. J. Culture media for use in the plate method of counting soil
bacteria. N. Y. Agr. Exp. Sta. Tech. Bui. 38. 1914.

27 Thornton, H. G. On the development of a standardized agar medium for
counting soil bacteria, with especial regard to the repression of spreading colonies.
Ann. Appl. Biol. 9: 241-274. 1922.

28 Cook, R. C. Quantitative media for the estimation of bacteria in soils.
Soil Sci. 1: 153-161. 1916.

16 PRINCIPLES OF SOIL MICROBIOLOGY

II. Lipman and Brown's synthetic agar:

Distilled water 1000 cc. Glucose 10 grams

Agar 20 grams MgS0 4 -7H 2 0.20 gram

Peptone 0.05 gram K 2 HP0 4 0.50 gram

III. Albumin agar:

Distilled water 1000 cc. MgS0 4 -7H 2 0.20 gram

Agar 15.00 grams K 2 HP0 4 0.50 gram

Powdered egg al- Fe 2 (S0 4 ) 3 trace

bumin 0.25 gram

Glucose 1.00 gram

The albumin is suspended in 5 to 10 cc. of water, then 1.0 cc. of 0.12V NaOH
solution is added, so as to convert it into sodium albuminate; the albuminate is
added only to the filtered medium.

IV. Casein agar. Same as medium III, only 1.0 gram of purified casein is
used in place of the albumin. The casein is dissolved in 8 cc. of 0.12V NaOH.

V. Asparaginate agar:

Distilled water 1000 cc. NH 4 H 2 P0 4 1.5 grams

Agar 12.0 grams CaCl 2 0.1 gram

Sodium asparaginate. 1.0 gram KC1 0.1 gram

Glucose l.Ogram FeCl 3 trace

MgS0 4 -7H 2 0.2 gram

10 cc. of 1.02V NaOH solution added per liter to bring the desired reaction.

VI. Asparagine-mannite agar:

Distilled water 1000 cc. NaCl 0.1 gram

Agar 15 grams FeCl 3 0.002 gram

K2HPO4 l.Ogram KN0 3 0.5 gram

MgS0 4 -7H 2 0.2 gram Asparagine 0.5 gram

CaCl 2 0.1 gram Mannite l.Ogram

The mannite is added after the agar and other constituents have been dis-
solved and medium filtered. The reaction is adjusted to pH 7.4.

VII. Urea ammonium nitrate agar:

Distilled water 1000 cc. Glucose 10.0 grams

Agar 15.0 grams Urea 0.05 gram

K 2 HP0 4 0.5 gram Ammonium nitrate. . . 0.1 gram

MgS0 4 -7H 2 0.2gram Fe 2 (S0 4 ) 3 trace

Reaction is about pH 7.0.

In addition to these media, soil extract and tap water gelatin recommended
by Conn and used chiefly for qualitative purposes can also be mentioned:

VIII. Tap water gelatin 29

Tap water 1000 cc.

Gelatin (Gold Label) 200 grams

These media are about equally favorable to the development of
aerobic, heterotrophic bacteria, deriving their nitrogen from inorganic

19 Medium is clarified by means of white of egg; reaction is adjusted to 0.5 per
cent normal acid to phenolphthalein, which requires 20-30 cc. 1.0 N NaOH; when
Bacto-gelatin is used, only 10 oc. of alkali is required and no clarification.

NUMBERS OF MICROORGANISMS

17

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or organic compounds, and of actinomyces, but give somewhat different
results for different soils, as shown by Conn. Egg-albumin agar and
especially soil extract agar were found by others 30 to give higher and
more uniform results than other media.

In preparing the media, the constituents including the agar or gelatin
are dissolved in boiling water: after filtration through cotton, the more
decomposable constituents are added, as in the case of urea, ammonium
nitrate and glucose in the urea nitrate agar, albumin and casein solu-
tions and the sodium asparaginate as well as the glucose, in the cor-
responding media, so as to avoid any possible effect of preliminary
heating on these substances.

The reaction of the media is an important factor. Its adjustment to
a definite point of titration is practically valueless in media, where the
buffer content is variable. 31 A slight acidity has usually been found to
be the most favorable reaction for the development of the majority of
soil bacteria. When determined by the hydrogen-ion concentration,
colorimetrically or electrometrically, this reaction was found to be pH
6.5 (albumin and casein media) to pH 7.0 (asparaginate agar media).

The media are placed in 10-cc. portions in test tubes, or in 100-cc. to
200-cc. portions in Erlenmeyer flasks. These are plugged with cotton
and sterilized in the autoclave, at 15 pounds pressure, for 15 to 20 minutes.
A soil may contain 5 to 30 million bacteria (including actinomyces)
per gram and only 50,000 fungi or less. The final dilution used for the
determination of bacteria would be 100,000 or 200,000. Some of the
plates will have no fungi at all, while others may have one, two or more
colonies. In this case the proper dilution for the determination of
fungi would be 1000, but there will be so many bacteria on the plate
that most of the fungi may actually fail to develop or make only a
scant growth. A medium should be used which allows only the develop-
ment of fungi. Use is made of the fact that the majority of fungi can
stand greater degrees of acidity than the bacteria and actinomyces
For qualitative purposes and for pure culture study, any medium favor-
able for the development of fungi, such as raisin extract (60 grams of
raisins heated at 60° to 75°C. in 1000 cc. of tap water, filtered), which is

30 Smith, N. R., and Worden, S. Plate counts of soil microorganisms. Jour.
Agr. Res. 31: 501-517. 1925.

31 Clark, W. M. The "reaction" of bacteriologic culture media. Jour. Inf.
Dis., 17: 109. 1915. Clark, M. W. The determination of hydrogen ions. 2d
Ed. The Williams & Wilkins Co., Baltimore. 1922; Gillespie, L. J. Colori-
metric determination of hydrogen-ion concentration without buffer mixtures,
with especial reference to soils. Soil Sci. 9: 115-136. 1920.

NUMBERS OF MICROORGANISMS 19

already acid in reaction, or a medium to which enough lactic acid is
added to bring the reaction to pH 4.0. Because of the high acidity,
25 gm. of agar are required per liter. For quantitative purposes,
however, a synthetic medium is always to be preferred : 32

Glucose 10 grams MgS0 4 -7H 2 0.5 gram

Peptone 5 grams Distilled water 1000 cc.

KH2PO4 1 gram

The ingredients are dissolved by boiling and enough normal acid (H2SO4 or
H3PO4) is added to bring the reaction to pH 3.6 to 3.8. This will require from
6 to 7 cc. of normal acid per liter of medium. Twenty-five to 30 grams of agar
ara added and dissolved by boiling; the medium is then filtered, tubed and ster-
ilized as usual. The final reaction of the medium should be pH 4.0.

For soils to which an excess Of organic matter has recently been added, the
dilution used for the determination of fungi is usually from one hundredth to
one-tenth of the dilution used for the determination of numbers of bacteria.

All the necessary glassware and water blanks should be prepared
and sterilized before the soil samples are taken. Three or four
pipettes and the same number of water blanks are required for every
soil sample. Ninety-nine or 90 cc. portions of tap water, depending
on the fact whether 1-cc. or 10-cc. pipettes are employed for making
the dilutions, are placed in the flasks which are then plugged with
cotton. The glassware (including the Petri dishes) is sterilized in
the hot air sterilizer for two hours at 160°C, while the water blanks
are sterilized in the autoclave, at 15 pounds pressure for 15 or 20
minutes. The sampling bottles may be sterilized either with the
other glassware or with the water blanks.

Sampling of soil. It has been shown both by cultural studies 18 and
by the use of the microscope 33 that a given soil will contain at a given
time, in a given soil layer, a uniform bacterial flora. Samples taken
under the same experimental conditions will, therefore, be the same bac-
teriologically, while any change in soil type and treatment will result
in a change in bacterial relations. When comparing different soils,
samples taken from the same depth should be compared. The varia-
bility of a soil itself, even as to physical and chemical factors, is so
great, however, that it is not surprising that such a sensitive indicator as
bacterial numbers and activities should also vary. To meet the factor
of variation, the common practice has been to use composite samples,

32 Waksman, 8. A. A method for counting the number of fungi in the soil.
Jour. Pact. 7: 339-341. 1922.

33 Winogradsky, 1925 (p. 7).

20 PRINCIPLES OF SOIL MICROBIOLOGY

taken from a thorough mixture of several representative borings to the
same depth in various parts of the field. This may be sufficient, if the
probable error involved in the determination is known. A study of the
variability of the soil in relation to the numbers of bacteria, as deter-
mined by the plate method, was found 34 to yield in one particular plot
eight to twenty-five millions per gram of soil. These results were based
on fifty-one samples of soil taken from one-twentieth of an acre; the
samples were taken at intervals of 5 by 8 feet. The numbers of bacteria
of practically a whole series of plots variously treated and where definite
bacteriological differences have been established were found to fall
within that range of numbers. This would make any results obtained
from a determination of numbers of bacteria based upon a single sample
practically valueless. Before any definite conclusions can be drawn
from a determination of bacterial activities in the soil, it is advisable to
study the variability of the particular soil. This is done by taking a
large number of samples from a particular field and working out the
probable error of the particular variable which should not be greater
than Em = 2.5 per cent.

For practical purposes, five composite soil samples each consisting
of three to four borings taken from different parts of the field will give
good results not only in the study of numbers of microorganisms but
also in the study of specific physiological activities of soil bacteria.

In taking the samples, a small amount of soil (j inch) is scraped away from
the surface by means of a spatula. The samples are taken by means of sampling
tubes or augers, carefully cleaned previously, to a depth of 6 to 6| inches, unless
a study is made of the distribution of microorganisms at different depths. In
that case a ditch may be made 3 feet long by 1 foot wide and, after scraping off the
surface soil, at the desired depth, by means of a spatula, the samples are taken;
small metallic sampling tubes (about 1 inch in diameter) made of iron or copper,
with sharp ends may also be used for this purpose.

Care is taken not to contaminate the soil samples with other soil or
by exposing them too long to the atmosphere. However, no absolute
sterility in the process of sampling is required, since the numbers of
microorganisms in the soil are very large in comparison with any possible
contamination from a brief exposure. The samples are placed in
sterile sampling bottles and brought to the laboratory as quickly as
possible. A rapid change was found 35 to take place in the numbers of

34 Waksman, S. A. Microbiological analysis of soil as an index of soil fer-
tility. I. The mathematical interpretation of numbers of microorganisms in the
soil. SoilSci. 14: 81-101. 1922.

88 Frankel, 1887 (p. 12).

NUMBERS OF MICROORGANISMS 21

soil microorganisms when the soil is kept for a short time in the labora-
tory. Some investigators 36 found a strong decrease in numbers, on
keeping the soil sample for any length of time. An increase can be
expected in soils, in which the activities of microorganisms have been
kept in check, as in the case of dry soils moistened, soils treated
with gaseous disinfectants, samples from low depths, frozen soils in
the process of thawing off. In the case of normal soils, a decrease may
be expected; Hiltner and Stormer recorded a change in the numbers
of bacteria from 7,519,000 to 2,087,000 in sixteen days and from
8,280,000 to 7,016,000 in six days.

The bacteriological analysis should, therefore, be made at once, or
as soon as the soils can be conveniently brought into the laboratory.

Treatment of soil samples and preparation of plates.

Before taking out portions of the samples for analysis, each sample is thoroughly
mixed by means of a clean spatula. When many small stones are present, the
sample is first sieved through a clean (sterile if possible) 2-mm. sieve. A definite
portion of soil is transferred by any convenient method into an Erlenmeyer flask
containing a definite quantity of sterile tap water, giving the first dilution. It is
preferable to have the first dilution 1:10 or 1:20, i.e., to use ten or twenty times
as much sterile water as soil taken. To give an initial dilution of 1:10, 10 gm. of
soil is added to 100 cc. of water. 37 If the soil contains considerable organic
matter, it may be advisable to triturate it first in a mortar with a little of the
diluting liquid. The weight of the soil is either included in the total volume of
the first dilution (10 grams of soil with sterile tap water to make 100 cc. volume)
or not (10 grams of soil added to 100 cc. of water).

The mixture of soil and water is shaken for five minutes. This results in
maximum counts, shorter periods being insufficient to separate all the bacteria
from the soil: longer periods of shaking have a destructive influence upon the
bacterial numbers. The soil should not be allowed to be too long in contact with
the water of dilution, otherwise an injurious effect will take place which is prob-
ably due to the plasmolytic action of the liquid. This leads to a rapid decrease
in the number of microorganisms affecting the vegetative cells first. An injurious
effect will occur only after 3-4 hours. 38

In preparing the further dilutions, the contents of the flask from which the
suspension is withdrawn should be kept in motion. The further dilutions can
be made up on the basis of 1:10 or to 1:100. It has been suggested 37,39 to limit
the further dilutions also to 1:10, i.e., each higher dilution should be made by

36 Hiltner and Stormer, 1903 (p. 12).

37 Wyant, Z. N. A comparison of the technic recommended by various authors
for quantitative bacteriological analysis of soil. Soil Sci. 11: 295-303. 1921.

38 Engberding, 1909 (p. 14).

39 Noyes, H. A., and Voigt, E. A technic for a bacteriologmarJr^^amipation of
soils. Proc. Ind. Acad. Sci. 1916, 272-301. /\y y ' */y\

L LIB R A R Y 2 J

22 PRINCIPLES OF SOIL MICROBIOLOGY

taking 10 cc. of the lower dilution and 90 cc. of the sterile diluting fluid. How-
ever, even by making higher dilutions such as 1:50 or 1:100 in each case, reliable
results are obtained.

The amount of soil used in making the first dilution, and the various
subsequent dilutions are not of primary importance, if care is taken in
the technic and in the calculations. By using for the first dilution 0. 1204
gram of soil, Hiltner and Stormer found 9,500,000 bacteria in 1 gram
of moist soil and, by using 6.7841 grams of the same soil, 9,400,000
bacteria were found in 1 gram of the same soil. In another soil, they
found, by using 0.5862 and 21.1820 gram portions of soil, counts of
7,750,000 and 7,700,000 respectively per 1 gram of soil. The higher the
dilution, the greater is the probable error of the results. The final dilu-
tion should be such as to allow between 40 and 200 colonies to develop
on the plate, in the case of bacteria (and actinomyces) . 40 The number of
fungus colonies allowed per plate on the special acid medium should be
20 to 100. This will necessitate that the soil should be diluted, in the
counting of fungi, only to about 500 to 2000, so that the highest dilution
is only iiU of that employed for the total bacterial numbers for the
same soil, in the case of productive soils. For soils very rich in organic
matter or for highly acid soils, the ratio between the highest dilution for
fungi and bacteria should be 1 : 50 or 1 : 10.

Sterile tap water should be used for making the dilutions. Distilled
water has an injurious effect (plasmolysis) . Salt solution or nutrient
medium have no advantage over ordinary tap water. Instead of the
usual shaking methods, a method has been suggested 41 for the disintegra-
tion and dispersion of the soil particles, by means of which plate counts
are supposed to give results comparable with direct counts. The use
of a deflocculating agent and protective colloid combined with long
vibration of the suspension were recommended. However, conditions
were made so as to favor bacterial development and the method cannot
be recommended without further study.

The plates are prepared from 1 cc. of the final dilution in a regular
bacteriological way. The number of plates to be used for each soil
sample is important. The number of colonies varies greatly with
the different plates and results based on averages of two or three
plates, without allowing for variability, makes them almost worthless.
Hiltner and Stormer recognized this fact and used four and sometimes

40 Breed, R. S., and Dotterer, W. D. The number of colonies allowable on
satisfactory agar plates. N. Y. Agr. Exp. Sta. Tech. Bui. 53. 1916.
"Whittles, 1923-24 (p. 7).

NUMBERS OF MICROORGANISMS 23

eight plates. The average error obtained was 8 to 10 per cent, in some
cases even 15 per cent, in other words, the difference between the ex-
treme variations was equivalent to ^ of the mean value. Variations
equal to one-third of the actual counts have been observed. 42 The
variability may be somewhat decreased by very careful technic, but
not reduced to a small error.

Incubation of plates and counting of organisms. The plates are incubated at a
constant temperature, preferably 25° to 27°C. for agar media, while gelatin
media are incubated at 18°C. The plates for the determination of fungi are
incubated for 4S to 72 hours ; a shorter period of time is insufficient for the develop-
ment of all the colonies, while a longer period favors rapid overgrowth of some
fungi. The colonies are counted after 48 and again after 72 hours. The bacterial
plates are incubated for seven to twelve days. A shorter period was found 43
to allow only an insufficient development of the microorganisms. When the plates
are incubated only two or three days not all the organisms develop into visible
colonies, especially the slow growing non-spore forming bacteria and actino-
myces. Hiltner and Stormer and others also found an incubation period of seven
days to be most favorable.

Lower temperatures require longer incubation periods, so that, at room temper-
ature, the plates should be incubated for fourteen days to give the maximum
development of microorganisms, while, at 25°, seven days are sufficient. Higher
temperatures, particularly above 30°C, have an injurious action upon the de-
velopment of certain soil organisms; the medium in the plate also dries out rather
rapidly.

At the end of the incubation period, all colonies on the plates are counted.
Where only 40 to 200 colonies are allowed per plate, it is sufficient to make with
a glass pencil two or three cross lines over the plate and count all the colonies with
the naked eye. But if, for one reason or another (pure culture study, for ex-
ample), a lower dilution is used and the number of colonies exceeds 200 (a shorter
incubation period may then have to be employed) the counting plate has to be
utilized. In that case, of course, one has to depend on the manufacturer for the
accuracy of divisions on the plate. A large enough number of sections should be
counted so as to reduce the error involved in the determinations to a minimum.
All the colonies, except the fungi, are counted and recorded as the total number
of organisms developing on the plate, where no separate count of actinomyces
is desired. Otherwise, after all the colonies have been counted, the actinomyces
are determined separately. It is advisable to have recourse to the microscope for
the examination of all the doubtful colonies, which are then marked off with a
glass pencil. The plates may also be incubated a few days (4 to 7) longer and
actinomyces colonies counted. The number of actinomyces may then be sub-
tracted from the "total number of organisms" to give the number of true bacteria.

42 Remy, Th. Bodenbakteriologische Studien. Centrbl. Bakt. II, 8: 657-662,
728-735. 1902.

43 Conn, H. J. Soil flora studies. I. Methods best adapted to the study of the
microscopic soil flora. N. Y. Agr. Exp. Sta. Tech. Bui. 57. 1917.

24 PRINCIPLES OF SOIL MICROBIOLOGY

The total number of organisms developing on the plate is recorded
either on the basis of moist soil, giving thereby the moisture content of
the soil, or on the basis of dry soil. The moisture is determined by
drying 10 grams of soil (50 or 100 grams may also be used, especially
in the case of non-homogeneous soil, like peat) at 100°C. to constant
weight. If N is the total number of organisms developing on the plate
from a given soil, a the average number for each plate, u the amount
of dilution, x the moisture content of the soil, then,

a • u • 100

N =

100 - x

Mathematical interpretation of results. The determination of numbers
of microorganisms by the plate method involves a number of manipula-
tions, each of which involves a certain error. The final error is a re-
sultant of all these errors. The constant errors should be eliminated,
but the occasional errors depend on the "Law of Chance" and these
tend to counterbalance one another rather than to fall in one direction.
A study of the variability of the number of bacteria, actinomyces, and
fungi on their respective media is given in table 2. The results were
calculated as follows:

If n plates are used for making a quantitative bacteriological count
of a given soil, a the number of colonies developing on the plates,
while z n is the most probable value obtained from counting n-plates,
or the arithmetic mean value, then we have

2 a

Zn =

n

In other words, the mean (z n ) is found by dividing the sum of all the
determinations by the number of determinations.

The following formulae are commonly used for calculating the average
deviation and most probable error of the determination.

a a • + - J S (Zn - a) 2
Average deviation, a = \

j n — 1

, ,, /S (Zn - a) 2

Most probable error = ± 0.6745 -4/

C.V. =

n(n — 1)

the average deviation X 100
mean

most probable error X 100

Em =

mean

NUMBERS OF MICROORGANISMS

25

TABLE 2
Numbers of microorganisms in the soil, as shown by the plate method*

BACTERIA AND ACTINOMYCES

64

96
81
53
96
69
72
62
69
77

106
93

98
87
91

Colonies

94

79

84

83

73

103

61

98

99

76

83

76

67

112

77

101

65

84

91

56

106
101
89
84
116
76
82
93
76
84

52
78
61
93
106

Total = 4173

Mean = 83.46 ± 1.5
a = 15.69 ± 1.07
C.V.= 18. 8 ±1.27 per

cent
A3 = ±2.22
Em = 1.8 per cent

ACTINOMYCES

Colonies

11

10
9

17
9

15
7

11

13
10

11

15

13
12

7

11
9
9

12
6

7
13
14
12
10
12
13
15
12
11

14
16
13
12
14
12
11

9
15

9

4
17
11
13
21

Total = 565

Mean = 11.3 ± 0.30
ff = 3.16 ± 0.20
C.V.= 27.96 ±1 89 per

cent
Az = ±0.45
Em = 2.7 per cent

Colonies

'3

6
3
5
2
3
4
2
3
2

4
3
3
5
1
1
1
1

1

2
1
1
1

2

Total = 138

Mean = 2.76 ±0.16
a- = 1.73 ±0.11
C.V.= 62.67 ±4.23 per

cent
Az = ±0.245
Em — 5.8 per cent

* In each group of 10 figures the number of colonies on each of 10 plates for each
soil sample is shown.

Approximately two-thirds of the determinations are expected to lie on
both sides of the mean, within that deviation.

26 PRINCIPLES OF SOIL MICROBIOLOGY

The probable error of the standard deviation is obtained from the

O"

formula ± 0.6745 /— • The coefficient of variability (C.V.) is the
V2n

percentage ratio of the standard deviation (<r) from the mean. The

C.V.
probable error of the coefficient of variability = ±0.6745 ,-■-• By the

use of formulae for the calculations of the errors of observation, the
error of the mean arithmetical value of the plate counts can be deter-
mined. 44

By taking several representative soil samples and by using several
plates, the error can be reduced to a minimum. A combination of five
soil samples and ten plates, for each soil to be tested, will reduce the
error for bacterial numbers to 1.8 per cent and for actinomyces to 2.7
per cent. 45 The same is true of fungi; when a low dilution (1000) is
combined with an acid medium, and 5 to 10 plates are used for each
sample, the error may be reduced to 2 per cent.

It is of interest to note here that in order to determine accurately the
average value of hygroscopic moisture in the soil, Floess 46 found that
fifty soil samples, taken at definite distances in the field, are required.
The probable error was calculated from the formula :

[v] • 0.845
r =

■\/n (n - 1)

Where [v] is the sum of all individual variations from the mean and n
the number of observations.

Under ideal conditions, the bacterial counts on parallel plates will
vary in a manner similar to samples from a Poisson series. 47 The
accuracy can thus be determined with precision and the mean count of a
number of plates is a direct measure of the bacterial population capable
of developing on the plate. Agreement with the theoretical distribu-
tion may be tested by means of the index of dispersion.

44 Davenport, C. B. Statistical methods. Wiley, New York. 1914. Barlow,
P. Tables of squares, cubes, square roots, cube roots, reciprocals. Spon.
London. 1914.

45 Waksman, 1922 (p. 20).

46 Floess, R. Die Hygroskopizitatsbestimmung, ein Masstab zur Bonitierung
des Ackerbodens. Landw. Jahrb. 42: 255-289. 1912.

47 Fischer, R. A., Thornton, H. G., and Mackenzie, W. A. The accuracy of the
plating method of estimating the density of bacterial populations. Ann. Appl.
Biol. 9: 325-359. 1922.

NUMBERS OF MICROORGANISMS 27

X 2 = = S (x - x) 2 ,

X

where x is the mean and x any individual number of colonies counted
on a plate; S stands for summation and X 2 as the index of variability.

With a carefully improved technic, an accurate conformity with the
theoretical distribution can be attained even with a mixed bacterial
flora of the soil. Any significant departure from the theoretical dis-
tribution is a sign that the mean may be wholly unreliable.

Co?nparison of plate and microscopic methods. By comparing the
results obtained by the microscopic and plate methods of determining
the numbers of microorganisms in the soil, Conn 48 came to the conclu-
sion that if an organism that can grow well on the plate is present in a
soil, the plate count will be nearly as high or even higher than the micro-
scopic count. In the case of large-sized bacteria, like Bac. cereus, the
microscopic count was found by Conn to be higher than the plate count ;
in the case of small bacteria, like Bact. fluorescens, or the orange-lique-
fying type, which are easily overlooked under the microscope, the plate
count is considerably higher. For the determination of the aerobic,
heterotrophic, non-nitrogen fixing bacteria in the soil, which develop
readily on the plate, the latter method is as satisfactory as the micro-
scopic method. A number of bacteria, like the cellulose-decomposing
forms, some urea bacteria, and others, even among the aerobic hetero-
trophic organisms, are not capable of developing on the common plate,
while some nitrogen-fixing bacteria may develop. The distinct differ-
ence between plate and microscopic counts of bacteria in normal soil
is due not only to the organisms which are not capable of growing on
common nutrient media, but also to the fact that a large number of
cells, even of the organisms capable of growing on the common plate,
do not develop into colonies. The actual number of bacteria in the
soil is probably five to twenty and even more times as high as the
counts obtained by the plate method.

For the present, however, the plate method is the most satisfactory
one for determining the relative abundance of microorganisms (bac-
teria, actinomyces and fungi) in the soil. For determining the num-
bers of protozoa and specific physiological groups of bacteria, the dilu-
tion method still remains the most satisfactory. In the case of actino-
myces and especially fungi, a colony on the plate represents either a
spore or a piece of mycelium in the soil; since the spore is only the

48 Conn, 1918 (p. 7).

28 PRINCIPLES OF SOIL MICROBIOLOGY

reproductive stage, while the mycelium is the active stage of the
organism, and since some fungi, like the Mucoraceae, produce a myce-
lium which does not readily break up into fragments, the results ob-
tained by plate count may be far from representing the actual abun-
dance of the particular organisms in the soil.

Numbers of bacteria in the soil. In view of the fact that the methods
used in the past for the determination of numbers of bacteria in the soil
varied greatly and most of the media were not synthetic in composition,
the results cannot be readily compared. However, certain funda-
mental facts have been established, which throw interesting light upon
the nature of the soil population. By the use of the microscopic method
Conn found, in one gram of soil, 140 to 390 millions of small rod-shaped
bacteria less than 0.5 micron in diameter, 1.5 to 12 million rods 0.5
to 0.8 micron in diameter and 500,000 to 5,000,000 spores per 1 gram
of soil. Very few of the large rods (over 1 micron in diameter) were
found in all the soil samples. In manured soils, the number of bacteria
were found to reach 800,000,000 per gram, as determined microscopically.
Still larger numbers were reported by others (p. 11). On the other
hand, by the use of nutrient agar plate, Adametz 49 found 320,000 to
500,000 bacteria per gram of sandy soil, and 360,000 to 600,000 in loamy
soils. The numbers of bacteria in the soil obtained by the numerous
investigators fall somewhere between these two sets of figures. These
tremendous differences and variable results led Lohnis 50 to the conclu-
sion that a determination of bacterial numbers in the soil is worthless
as an attempt of interpreting soil phenomena. However, when the
results are considered critically and compared carefully, it is found
that the information obtained from the study of numbers of micro-
organisms gives not only an interesting insight into the microbiological
population of the soil, but also throws light on soil fertility, particularly
when this information is combined with that obtained from the study of
physiological activities of soil microorganisms (p. 711).

In general the numbers of bacteria, as determined by the plate
method, are found to range in normal soils well supplied with organic
matter from 2 to 200 millions per gram, as determined by the plate
method. These numbers vary with the soil type, soil treatment,
season of year, depth, moisture content and various environmental

49 Adametz. Untersuchungen iiber die niederen Pilze der Ackerkrume. Diss.
Leipzig. 1886.

10 Lohnis, F. Handbuch der landwirtschaftlichen Bakteriologie. Berlin,
1910, p. 513.

NUMBERS OF MICROORGANISMS 29

conditions. No two soils, even from the same locality can be com-
pared on the basis of mere bacterial numbers without a detailed
knowledge of the physical and chemical condition of soil, treatment,
etc. In some soils, like very poor sandy soils, very acid peat soils
and abnormal acid or alkaline soils, the number of bacteria may be
much below 2,000,000 per gram. The sandy soils of the Pine-
barrens of New Jersey, for example, contain only 25,000 to 100,000
bacteria per gram, while certain acid peat and acid forest soils may be
nearly free from bacteria capable of developing on the common agar or
gelatin plate. Very heavily manured soils, such as greenhouse soils
or market garden soils, particularly at the time of active decomposition
of the organic matter, may contain over 200,000,000 bacteria per gram,
even as shown by the plate method.

Bacterial numbers in manure. Manure consists of (1) solid excreta,
(2) straw, hay or peat litter, and (3) urine. Solid excreta are very rich
in bacteria. The bacterial numbers in feces were found 51 to be very
variable; 1 gram of fresh human feces may contain 18,000,000,000
bacteria. Dry cow manure was shown 52 to contain, by weight, 9 to
20 per cent bacteria. One gram of fresh cow manure (17.65 per cent
dry weight) should thus contain 18 to 40 billions bacteria, the larger
number of which consists of dead cells. However, by the plate method,
only 40 to 70 millions bacteria per gram of cattle manure and 100 to 150
millions per gram of horse manure were obtained. By the use of the
gravimetric method, Lissauer 53 estimated that 4.26 to 15.67 per cent of
the feces of man (on the average 9 per cent), 3.54 to 9.08 of dog, 0.41
to 1.31 of rabbit and 14.73 to 18.75 per cent of the feces of cattle con-
sisted of bacteria; the kind of food (vegetable or animal) influenced the
numbers. Since 1 mgm. of dry bacteria contains 4,000,000,000 cells,
1 mgm. of cow feces would contain 63 to 80 millions of bacteria. By the
plate method, manure of stall-fed cattle was found 54 to contain 1 to 120
million bacteria per gram, while that of cattle on pasture contained only

61 Matzuschita, T. Untersuchungen liber die Mikroorganismen des men-
schlichen Kotes. Arch. Hyg. 41: 210-255. 1901.

62 Stoklasa, J. tlber die Wirkung des Stallmistes. Ztschr. landw. Versuch.
Osterreich. 10: 440. 1907.

63 Lissauer, M. Uber den Bakteriengehalt menschlicher und tierischer Faces.
Arch. Hyg. 58: 136-149. 1906.

64 Gruber, Th. Die Bakterienflora von Runkelriiben, Steckruben, Karotten,
von Milch wahrend der Stallf iitterung und des Weideganges einschlieszlich der
in Streu, Gras und Kot vorkommenden Mikroorganismen und deren Mengen-
vehaltnisse in den 4 letzten Medien. Centrbl. Bakt. Abt. II, 22: 401-416. 1909.

30 PRINCIPLES OF SOIL MICROBIOLOGY

1 to 4 million. One billion bacteria were demonstrated 55 in 1 cc. of the
intestinal contents of the ox.

However, many of the earlier investigators recorded too low
numbers, due to faulty technic. In the more recent studies 56
12,000,000,000 living microorganisms were recorded per 1 gram of
compost of manure and straw and even these figures were too low, due
to the fact that not all groups of organisms were counted. The feces
of white rats was shown 57 to consist of 33 to 42 per cent bacteria by
weight, although no attempt was made to differentiate between the
living and dead cells.

The bacterial content of straw also varies greatly. Seventy-four
thousand to 11,640,000 bacteria were found 58 per gram of straw and a
decrease was observed as a result of drying and preservation under clean
surroundings. The numbers of bacteria per gram of hay may vary
between 10 and 400 millions per gram. 59 Figures varying from 3.6 to
600 millions of microorganisms per gram of straw were also reported.
These organisms consist of non-spore formers (Bad. herbicola, Bad.
giintheri, Bad. acidi ladici, Bad. fluorescens), spore-formers, butyric
acid bacteria, cocci, actinomyces (2 to 83 per cent of flora) and a few
fungi. 00

Urine is practically sterile, when it leaves the healthy body, but,
on exposure, bacteria begin to multiply very rapidly, and soon an
abundant flora consisting of bacteria and protozoa may appear.

The number of bacteria in stable manure will vary greatly, depending
on its composition, the amount of solid excreta, and the degree of decom-
position. The numbers will also depend of course upon the method
used for their estimation, plate, dilution and microscopic methods
giving different results. Fresh manure is very rich in Bad. coli, which

55 Huttermann, W. Beitriige zur Kenntnis der Bakterienflora im normalen
Darmtraktus des Rindes. Diss. Bern. 1905; Koch's Jahresb. Giirungs. 16: 402.
1905.

56 Lohnis, F., and Smith, J. H. Fiihl. landw. Ztg. 63: 153. 1914.

67 Osborne, T. B., and Mendel, L. B. Jour. Biol. Chem. 18: 177. 1914.

68 Hoffmann, F. Wie grosz is die Zahl der Mikroorganismen auf dem Getreide
unter verschiedenen Bedingungen. Woch. Brau. p. 1153; Koch's Jahresber.
Garungs. 7: 67-68. 1896. Esten, W. M., and Mason, C. J. Sources of bacteria
in milk. Storrs (Conn.) Agr. Exp. Sta. Bui. 51. 1908.

50 Duggeli, M. Beitrag zur Kenntnis der Selbsterhitzung des Heues. Naturw.
Ztschr. Land. Forstw. 4: 473-492. 1906.

60 Kursteiner, R. Die Bakterienflora von frischen und benutzten Streumate-
rialien, mit besonderer Berucksichtigung ihrer Einwirkung auf Milch. Centrbl.
Bakt. 11,47: 1-191. 1916.

NUMBERS OF MICROORGANISMS

31

soon disappears in the composting of the manure. At first there is a
multiplication of the bacteria, soon followed by a reduction in numbers,
when the process of rotting of the manure is advanced. In manure
fourteen years old and greatly reduced in volume, 12.5 millions of
bacteria were found per gram (using manure extract-gelatin medium
for counting), while manure preserved with superphosphate, gypsum
and kainit contained 3.75 millions bacteria per gram. ei

Fig. 1. Upper curves show millions of bacteria per gram of dry soil, through-
out the year: good soil, poor soil; lower curves show: soil

moisture in per cent; atmospheric temperature; soil temperature;

shaded columns represent rainfall per week, in millimeters (from Conn).

Numbers of bacteria in the soil during different seasons of the year.
The season of the year cannot be considered as one single variable, so
that its influence upon bacterial numbers and activities could be studied.
The moisture content of the soil at the particular date of sampling,
kind of crop, stage of growth of crop, previous soil treatment, and many
other factors will influence the numbers and types of microorganisms
found in the soil. Few bacteria may be found in the soil at the end of a
long dry period. After the first rainfall, there is a rapid increase in

61 Lohnis, F., and Kuntze, W. Beitrage zur Kenntnis der Mikroflora des
Stalldilngers. Centrbl. Bakt. II, 20: 676-687. 1908.

32 PRINCIPLES OF SOIL MICROBIOLOGY

numbers, due to the fact that a continued dry period brings about a
phenomenon similar to partial sterilization of the soil and with the first
increase in moisture there is an increase of available organic and in-
organic materials, leading to a rapid increase in bacterial numbers.
Several investigators found larger bacterial numbers in the soil in sum-
mer than in winter, the maximum being reached in July and August.
In the spring of the year, with the increase in soil temperature, there is a
corresponding increase in bacterial numbers. 62 It was suggested, 38
however, that the water content of the soil is the most important factor
bearing upon bacterial numbers: cultivation increases the number by
increasing the water content. Engberding 38 was unable to demonstrate
any influence of temperature upon the numbers of bacteria in mineral
soils. This difference in results may be due to the difference in the
composition of the soil. The numbers of bacteria (and protozoa) were
even found to fluctuate from day to day. 63 Well-marked seasonal
changes in the soil population are superimposed on the daily varia-
tions in numbers. These changes are not directly influenced by tem-
perature or rainfall, but show a similarity to the seasonal fluctuations
recorded for many aquatic organisms.

Conn, 61 after a careful comparison of bacterial numbers in frozen
and unfrozen soil, came to the conclusion that the number of bacteria
in frozen soil is generally larger than in unfrozen soil, which is true not
only of cropped soil, but also of sod and fallow land. This increase in
bacterial numbers after freezing was believed not to be due to an increase
in moisture content, even though in an unfrozen condition the bacterial
numbers seemed to increase and decrease parallel to the moisture con-
tent of the soil. The increase in frozen soil seemed to be a result of
actual multiplication of the bacteria, rather than of a mere rise of the
organisms from lower depths brought about by mechanical forces
alone. Conn, therefore, suggested that there are two groups of bacteria
in the soil, "summer" and "winter" bacteria, which are active in the
respective periods. These results were at first confirmed. 65 The

62 Remy, 1902 (p. 23).

83 Cutler, D. W., Crump, L. M., and Sandon, H. A quantitative investigation
of the bacterial and protozoan population of the soil, with an account of the
protozoan fauna. Phil. Trans. Roy. Soc. London, B, 211: 317-350. 1922.

64 Conn, H. J. Bacteria in frozen soils. Centrbl. Bakt. II, 28:422-434.
1910; 32: 70-97. 1911; 42: 510-519. 1914; N. Y. Agr. Exp. Sta. Tech. Bui. 35.
1914.

66 Brown, P. E., and Smith, R. E. Bacteria at different depths of some typical
Iowa soils. Iowa Agr. Exp. Sta., Res. Bui. 8: 281-321. 1912.

NUMBERS OF MICROORGANISMS 33

theory was suggested that surface tension exerted by the soil particles
on the films of water, as well as the presence of salts in the water and the
concentration of salts, which may occur when the main body of water
begins to freeze, all cause the hygroscopic water in soils to remain un-
congealed, and consequently bacteria may live in it and multiply to a
comparatively large extent. Other results 66 also tended to indicate that
low temperatures may greatly increase the numbers of bacteria. To
explain these phenomena, the mechanical transportation of the bacteria
by the moisture coming up from below during heavy frost was sug-
gested. 67 It was later found, 68 however, that a slightly frozen condition
of the soil allowed bacterial development, but severe frosts produced a
checking action, the decrease being parallel with the depression of
temperature. No change in crop and plant remains took place in
Canada during the winter since the temperature of the soil goes down too
low. The occurrence of two maximum counts of bacteria observed 69
in Iowa soils during the year, on February 12 and June 19, with inter-
vening minimum counts, were used to prove the theory that tempera-
tures much below zero are necessary before the hygroscopic moisture
freezes and, until that occurred, a development of bacteria might be
expected. It may also be suggested that a slight freezing of the soil,
in modifying the colloidal condition of the soil may have the same stim-
ulating action as air drying, treating with disinfectants, etc., in other
words, shifting the soil equilibrium, so that a more rapid multiplication
of the bacteria may take place.

A more recent careful study of the influence of freezing upon soil
bacteria demonstrated that the increase recorded previously is not
due to an actual multiplication of the bacteria but rather to a breaking
up of the clumps of bacteria in the soil resulting in a larger number of
colonies developing on the plate. 70 The moisture content and rate of

66 Weber, G. G. A. Die Einwirkung der Kalte auf die Mikroorganismen und
ihre Tatigkeit im Boden. Diss. Jena, 88. 1912. (Centrbl. Bakt., II, 37: 113.
1912.)

67 Harder, E. G. The occurrence of bacteria in frozen soil. Bot. Gaz. 61:
363. 1916.

68 Vanderleck, J. Bacteria of frozen soils in Quebec. I and II. Trans. Roy.
Soc. Canada (Ser. Ill, Sec. IV), 11: 15. 1918; 12: 1. 1918.

69 Brown, P. E., and Halversen, W. V. Effect of seasonal conditions and
soil treatment on bacteria and molds in the soil. Iowa Agr. Exp. Sta., Res. Bui.
56: 251-275. 1919.

70 Vass, A. F. The influence of low temperature on soil bacteria. Cornell
Univ. Agr. Exp. Sta. Memoir 27. 1919.

34 PRINCIPLES OF SOIL MICROBIOLOGY

thaw are important factors, in determining the extent of the breaking
up process. The work of Lochhead 71 also tends to refute the idea that
there are in the soil two groups of bacteria, winter and summer forms.
Although the numbers of bacteria in frozen soil are high, there is no
phenomenal increase, as a result of freezing, while thawing of the soil
brought about a great increase in numbers. A typical winter flora is
absent and the bacteria are to be regarded as cold-enduring rather than
psychrophilic in the true sense. There was also no indication of a rise
of organisms from unfrozen levels to the frozen surface layers.

Distribution of bacteria at various soil depths. In humid soil, most of
the bacteria are concentrated in the upper 2 feet of soil and the highest
numbers are found just 1 to 3 inches below the surface. The germicidal
effect of the rays of the sun and the rapid evaporation of the moisture
tend to diminish the numbers right at the surface. Much fewer bac-
teria are present in the subsoil; in sandy soils, due to better aeration,
the numbers do not diminish as rapidly. Since most of the earlier work
was limited to the use of the plate method, the results are of necessity
too low.

Proskauer 72 was the first to point out the fact that the numbers of
bacteria in humid soils decrease with depth: at a depth of about one
meter the soil was found to be almost free from bacteria. Frankel 73
observed a decrease of bacterial numbers with depth of soil, from 90,000
to 300,000 at the surface, to 100 to 700 at a depth of 2.5 meters. The
change was not gradual, but sudden and irregular. The bacteria were
found to go deeper in cultivated than in uncultivated soils, but no in-
fluence of the crop upon the number of organisms could be demonstrated.
A decrease from 2,564,000 bacteria at the surface to none at a depth of
six feet was observed 74 in a stony soil, and from 524,500 at the surface
to 5,800 at a depth of 1.5 meters in a wet meadow soil. Clover soils
contained 6,000,000 bacteria at a depth of 20 cm. and 1,500,000 at 50
cm. depth. 75 A change from 8,000,000 bacteria per gram of soil at the
surface to sterility at a depth of one meter was also recorded. 76 The

71 Lochhead, A. G. Microbiological studies of frozen soil. Trans. Roy. Soc-
Canada. 18: 75-96. 1924; Soil Sci. 21: 225-232. 1926.

72 Proskauer, 1882 (p. 12).
"Frankel, 1887 (p. 12).

74 Reimers, J. tlber den Gehalt des Bodens in Bacteria. Ztschr. Hyg. 7:
319-346. 1889.

76 Caron, A. Landwirtschaftlich-bakteriologische Probleme. Landw. Vers.
Sta., 45: 401-418. 1895.

75 Stoklasa, J., and Earnest, A. tjber den Ursprung, die Menge und die
Bedeutung des Kohlendioxyds im Boden. Centrbl. Bakt. II, 14: 723-736. 1905.

NUMBERS OF MICROORGANISMS

35

numbers of bacteria usually increase from the surface to a depth of 4 to
6 inches; 77 this is followed by a decrease to a depth of 24 inches m humid
soils. The maximum numbers were found at six inches of depth; 78 in
some cases 79 the maximum was found at a depth of 4 inches, where the
greatest numbers of organisms are found followed by a more or less
regular drop. Soils under shade (orchard, forest, meadow) have the
highest numbers of bacteria at a depth of 1 inch, then the numbers

Numb era

10,000,000fy
9,000,00
8,OOO,OO0fX
7,000,000
6,000,000
5,000,000
4,000,000
3,000,000
2,000,000
1,000,000

Inches °
deplh-

.„_.._ Soil C.
Soil D.

Fig. 2. Numbers of bacteria at different depths of soil, on the average of
several determinations throughout the year: A, rich garden soil; B, cultivated
orchard soil; C, clay soil, under timothy; D, acid forest soil (from Waksman).

decrease with depth, while soils exposed to the sun have the highest
numbers at a depth of 4 inches. 80

77 Chester, F. D. The bacteriological analysis of soils. Del. Agr. Exp. Sta.
Bui. 65. 1904.

78 King, W. E., and Doryland, C. J. T. The influence of depth of cultivation
upon soil bacteria and their activities. Kans. Agr. Exp. Sta. Bui. 161: 211-242.
1909.

79 Brown and Smith, 1912 (p. 32).

80 Waksman, Soil Sci., 1 : 363-380. 1916.

36 PRINCIPLES OP SOIL MICROBIOLOGY

In the case of arid soils, the microorganisms are found to penetrate
much deeper into the subsoil layers, the activities of the organisms con-
tinuing in some cases undiminished through six feet of soil. The num-
bers of bacteria may even be greater at a depth of 24 inches than at 6
inches; 81 a wind-blown arid soil of Arizona, of a pH 8.6 to 9.0 and with an
average annual rainfall of 13 inches was found to contain 401 ,000 organisms
at a depth of 6 inches, and 1,898,500 at a depth of 12 inches and 916,500
at a depth of 24 inches. Fifty-two per cent of the organisms developing
on the plate were actinomyces. Better aeration and presence of organic
matter are no doubt largely responsible for this greater distribution of
microorganisms with depth of soil in arid regions. The favorable in-
fluence of irrigation upon bacterial numbers is marked not only at the
surface but also at lower depths, 82 as shown in table 3. Further informa-
tion on the influence of depth upon microorganisms and their activities
is given elsewhere (p. 48).

TABLE 3

Distribution of soil bacteria in different depths of soil in arid regions

DEPTH

IRRIGATED SOIL

DRY-FARM SOIL

1 foot

2 feet

3 feet

6,240,000
1,760,000
1,147,000

4,372,000
1,267,000
1,174,000

Numbers of specific physiological groups of bacteria in the soil. In
determining the numbers of specific morphological or physiological
groups of bacteria in the soil, the dilution method is largely employed,
as outlined above. 8384 The number obtained indicates a certain mini-
mum of cells of the particular organism, since often many of the cells
introduced into the specific medium may fail to develop. Hiltner and
Stormer found that a soil, which gave by the gelatin plate 1,270,000
bacteria, contained a much greater number of bacteria, when deter-
mined by the dilution method.

81 Lipman, C. B. The distribution and activities of bacteria in soils of the
arid region. Univ. Cal. Publ. Agr. Sci. 1: 7-20. 1912;4:113-120. 1919. Snow,
L. M. A comparative study of the bacterial flora of wind-blown soil. I. Arroyo
bank soil. Tucson, Arizona. Soil Sci. 21: 143-161. 1926.

82 Greaves, J. E. Agricultural bacteriology. Lea and Febiger. Phila., p.
164. 1922.

83 L6hnis, 1905 (p. 13).

84 Millard, W. A. Bacteriological tests in soil and dung. Centrbl. Bakt. II,
31: 502-507. 1911.

NUMBERS OF MICROORGANISMS

37

Although by the plate method the number of Azotobacter cells
present in the soil (when they are present at all) is rather small, amount-
ing to only a few hundreds (800 to 1000) 85 or a few thousands 86 per
gram, the direct microscopic examination reveals a great abundance of
these organisms in soils properly limed and buffered. Clostridium
pastorianum (Bac. amylobacter A. M. et Bred.) may be found in num-
bers ranging from 1 to 5 millions per gram of soil. In poorly buffered
and in acid soils, Azotobacter is absent altogether and its place is taken
by the Clostridium, as shown later.

For the determination of the numbers of bacteria belonging to the
genus Rhizobium, a medium containing 2 grams levulose, 0.06 gram
asparagine, 0.1 gram sodium citrate, 0.1 gram potassium citrate and 2
grams of agar in 100 parts of tap water may be used. 87 When the plates
are poured, 3 to 5 drops of normal solution of sodium carbonate are

TABLE 4
Numbers of physiological groups of bacteria in 1 gram of soil

I. Peptone decomposing
II. Urea decomposing. . .

III. Nitrifying

IV. Denitrifying

V. Nitrogen-fixing

RESULTS OF

RESULTS OF

RESULTS

LOHNIS, 83 AVER-

MILLARD, AVER-

OF HILTNER AND

AGE OF SUMMER

AGE OF MAXIMUM

STORM ER

AND WINTER

AND MINIMUM

VALUES

VALUES

3,750,000

4,375,000

75,000,000

50,000

50,000

27,500,000

7,000

5,000

100,000

50,000

50,000

162,500

25

388

2,500,000

added to 10 cc. of medium. Most bacteria and molds do not develop
on this medium; 50 to 8G per cent of the colonies are Rhizobia which
are small or punctiform, white, somewhat stiff and gummy. To con-
firm the diagnosis, the colonies are transferred to plates of nutrient
agar or plant extract (beans) agar. The numbers of Rhizobia in the
soil were found to run parallel with its fertility, and as many as 3 to 4

85 Truffaut, G., and Bezssonoff, H. Augmentation du nombre des Clostridium
Pastorianum (Winogradsky) dans les terres partiellement sterilisees par le sulfure
decalcium. Compt. Rend. Acad. Sci. 172: 1319-1324. 1921;173:868-870. 1921.
La Science du Sol. 1: 3-61. 1922.

86 Razoomov, A. S. Method of counting soil bacteria according to their physi-
ological groups (Russian). Trans. Institute of Fertilizers, No. 28, Moscow.
1925.

87 Greig-Smith, R. The determination of rhizobia in the soil. Centrbl. Bakt.
II, 34: 227-229. 1912.

38

PRINCIPLES OF SOIL MICROBIOLOGY

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NUMBERS OF MICROORGANISMS 39

million cells were found per gram of soil, comprising 0.4 to 6.75 per cent
of the total flora developing on the plate. Since Rhizobium was found
to fix 3 to 5.6 mgm. of nitrogen for 100 cc. of solution, it was believed
to be identical with Bad. radicicola.

The use of starch agar for determining the numbers of organisms in
the soil capable of utilizing starch has also been suggested. 89

A detailed report of the abundance of various physiological groups of
bacteria in different soils is given in table 5, based upon three deter-
minations made by Diigelli 88 in 1920, using two samples for each de-
termination.

Numbers of actinomyces in the soil. The synthetic media (p. 16)
best adapted for the growth of bacteria are also well adapted for the
growth of actinomyces, and their numbers can be determined on the
same plate used for bacterial numbers. If the reaction of the media
favorable for the study of numbers of the three groups of soil organisms
are compared, the optimum for actinomyces is found to be pH 7.0 to
7.5, for bacteria pH 6.5 to 7.0, for fungi pH 4.0. Of course, these are
not the optima for the growth of the particular organisms in pure
culture. The incubation period for counting actinomyces should be at
least 7 days and, if possible, 14 days, at 25° to 30°C.

The actinomyces are, next to the bacteria, the most abundant group
of organisms in the soil, as far as forms developing on the plate are
concerned. Since a colony arises from a conidium, a chain of conidia
or a piece of vegetative mycelium, larger numbers may not necessarily
indicate more abundant vegetative growth, but a larger abundance of
conidia.

Hiltner and Stormer observed an increase in the numbers of actino-
myces in the autumn, relative to the other groups of microorganisms,
due to the increase of the content in undecomposed organic matter in
the soil. They were found to form in the spring 20 per cent, in the fall
30 per cent, dropping in the winter to 13 per cent, of the total soil
microbial flora developing on the plate. The addition of stable manure,
due to its straw content, results in an increase in the numbers of
actinomyces.

88 Diiggeli, M. Die Bakterien des Waldbodens. Schweiz. Ztschr. f. Forstwes.
1923; Forschungen auf dem Gebiete der Bodenbakteriologie. Landw. Vortrage.
Verlag Huber, Frausenfeld. 1921. H. 3.

89 Hoffmann, C. A contribution to the subject of soil bacteriological analytical
methods. Centrbl. Bakt. II, 34: 386-388. 1912.

40

PRINCIPLES OF SOIL MICROBIOLOGY

Loam soils were found 90 to contain a higher number of actinomyces than
other soils, sandy soils coming last; a proportionate increase of these
organisms was found in the fall (27 to 35 per cent) over the spring (18 to
23 per cent), but no reduction was observed in the winter. Cultivation
of the soil effected a decrease in the numbers of actinomyces. These
organisms are also found abundantly in forest soils (24 to 27 per cent
of the flora) and on the roots of different plants, particularly grass and
legume roots, the upper cell layers of which died down.

The soil microflora developing on the plate may consist of as many as
40 per cent of actinomyces; the total number of these organisms was
found to be as high as 12 to 14 millions per gram of soil. 91,92 Sod soils
contain larger numbers of actinomyces than cultivated soils and it was

TABLE 6

Bacteria and actinomyces at various depths

Average of three New Jersey soils

BACTERIA

ACTINOMYCES

Numbers in 1
gram

Per cent

Numbers in 1
gram

Per cent

inches

1

7,340,000

90.8

743,000

9.2

4

5,300,000

85.0

933,000

15.0

8

2,710,000

81.6

612,000

18.4

12

950,000

79.9

239,000

20.1

20

259,000

51.3

246,000

48.7

30

124,000

34.6

240,000

65.6

suggested, therefore, that they may play an active part in the decom-
position of organic matter in the soil. 93 The numbers of actinomyces
decrease regularly with depth, but they increase in proportion to the
other microorganisms, so that at a depth of 30 inches they form 65. G
per cent of the total microbial flora developing on the agar plate. 94

90 Fousek, A. tlber die Rolle der Streptotricheen im Boden. Mitt. Landw.
Lehrkan. K. K. Hochschule f. Bodenk. Wien, 1: 217-244. 1913.

91 Conn, H. J. Distribution of bacteria in various 'soil types. Jour. Amer.
Soc. Agron. 5: 218-221. 1913.

92 Krainsky, A. Die Aktinomyceten und ihre Bedeutung in der Natur.
Centrbl. Bakt. II, 41: 649-688. 1914.

93 Conn, H. J. A possible function of actinomycetes in soil. Jour. Bact. 1:
197-207. 1916; N. Y. State Agr. Exp. Sta. Tech. Bui. 60.

94 Waksman, S. A., and Curtis, R. E. The actinomyces of the soil. Soil
Sci. 1: 99-134. 1916.

NUMBERS OF MICROORGANISMS

41

Similar results were obtained by other investigators. 95 It is not known
whether this is due to the greater resistance of these organisms to the
lack of oxygen, to the washing down of the conidia, or to some other
cause. Since these organisms are very sensitive to acidity and to an
excess of moisture, acid soils and water-logged soils contain a minimum
number of actinomyces; since they play an active part in the decom-
position of soil organic matter, heavy soils and soils rich in undecom-
posed organic matter, especially when well limed or buffered, contain

Noun- Occ.i Dec.19 Jan.7 Oanze, Feb.H Fetu? Mar2l Aprs Aprzz

Fig. 3. Variation of numbers of bacteria and actinomyces at different seasons
of the year and different depths of soil (from Lochhead).

high numbers of actinomyces, in comparison with the bacteria of the
same soil.

The addition of manure 96,97 as well as of different other forms of unde-
composed organic matter greatly stimulates the development of actino-
myces and may even result in an increase in their relative abundance, in

96 Lochhead, 1924 (p. 34).

96 Hiltner, L. Uber neuere Ergebnisse und Probleme auf dem Gebiete der
landwirtschaftlichen Bakteriologie. [ Jahrb. Ver. angew. Bot. for 1907, 5: 200-222.
1908.

97 Bright, J. W., and Conn, H. J. Ammonification of manure in soil. N. Y.
Agr. Exp. Sta. Tech. Bui. 67. 1919.

42

PRINCIPLES OF SOIL MICROBIOLOGY

comparison with the other organisms developing on the plate. Among
the other most important factors influencing the total and relative
abundance of actinomyces in the soil is the soil reaction; the less acid
the soil, the higher is the relative number of actinomyces. The in-
fluence of soil treatment upon the development of these organisms in
the soil is shown in table 7.

TABLE 7
Influence of soil treatment on the number of actinomyces in the soil

TREATMENT OF SOIL

ACTINOMYCES
PER GRAM

PER CENT OF

FLORA

DEVELOPING

ON PLATE

SOIL REACTION

PH

Unfertilized

1,150,000
2,410,000
1,520,000
2,920,000
370,000
2,520,000
2,530,000

27.7
31.6
22.7
24.5
12.1
26.5
25.0

4 6

Lime alone

64

Minerals*

5.5

Manure and minerals

5.4

Minerals and ammonium sulfate

Minerals, ammonium sulfate and lime . . .
Minerals and sodium nitrate

4.1

5.8
5.5

* 320 pounds of KC1 and 640 pounds acid phosphate per acre every year.

TABLE 8
Influence of liming and fertilization upon the numbers of fungi in the soil

SOIL TREATMENT

Unfertilized

Lime alone

Minerals

Minerals + lime

Manure and minerals

Manure, minerals and lime . .

NaN0 3 and minerals

(NH 4 ) 2 S0 4 and minerals

Manure, NaN0 3 and minerals

NUMBERS

PH

OF FUNGI PER

GRAM

4.8

65,500

6.4

16,900

5.6

45,600

6.4

26,300

5.6

91,000

7.0

29,400

5.8

45,900

4.6

107,900

5.8

87,600

Numbers of fungi in the soil. Fungi also exist in the soil both in the
form of vegetative mycelium and reproductive spores. Methods for
demonstrating the existence of specific forms in the soil are described
elsewhere (p. 239). There is no method yet devised for finding out how
much of the fungus material exists in the soil as mycelium or as spores ;
we neither have means of determining how many of the colonies develop-

NUMBERS OF MICROORGANISMS

43

ing on the plate are due to spores and how many to pieces of mycelium.
There is also no basis for comparing the relative abundance and potential
activity between the bacterial and fungus flora of the soil; for example,
1000 colonies of fungi may indicate a greater activity than 1,000,000
colonies of bacteria, when a certain process, such as cellulose or protein
decomposition is studied. However, if the 1000 fungus colonies repre-
sent inactive spores, the activity of the organism may be questioned.
With these limitations in mind and in view of the fact that most of the
determinations of numbers of fungi have been made by the use of high

Nwi4- Dec I Dec 19 Jan 7 Jon 23 Febu Feb 29 Mar^i Aprs April

Fig. 4. Variation of numbers of fungi at different seasons of the year and
different depths of soil (from Loehhead).

dilutions and of the bacterial agar plate, it is questionable to what extent
many of the results obtained in the past actually represent even relative
soil conditions. In general, the soil is found 98 to harbor between 30,000
and 900,000 fungi (spores and pieces of mycelium) per gram, depending
on the type of soil and treatment. Others" reported a range of 42,000

98 Waksman, S. A. Soil fungi and their activities. Soil Sci. 2: 103-155.
1916; Is there any fungus flora of the soil? Ibid. 3: 565-5S9. 1917; Waksman,
S. A. Influence of soil reaction upon the distribution of fungi in the soil. Ecol-
ogy, 6: 54-59. 1924.

89 Brown and Halversen. 1919 (p. 33).

44

PRINCIPLES OF SOIL MICROBIOLOGY

to 131,000 fungi per gram of soil when plated out on three different
media, with a ratio between the fungi and bacteria developing on the
plate as 1:40 or 1:50. However, whenever possible, these results
should be checked up by microscopic observations.

An acid medium similar to the one described above, combined with a
low dilution allows the determination, with a fair degree of accuracy, of

7A 7B

19A 4A

14 000

12.000

10.000

8.000

.000

2.000

Bacteria, thousands per gram
Fungi, hundreds per gram

-, — —_ — — — - -. Average yield, 1908-1921

Yield for 1921

Fig. 5. Influence of soil treatment upon the numbers of bacteria and fungi
in the soil: 7A and 7B, no fertilizer or manure; 4A, 19A, 19B, receiving only
mineral fertilizers (phosphate and KC1); 11A and 11B, ammonium sulfate and
minerals; 9A, sodium nitrate and minerals; 5A, stable manure and minerals;
the B plots limed in 1908, 1913, 1918 (after Waksman and de Rossi).

the relative abundance of fungi in the soil. The more acid the soil is,
the greater is the number of fungi relative to the number of the other
soil microorganisms. Figure 4 gives the changes in the number of
fungi during the year and at different depths and fig. 5 shows the in-
fluence of soil treatment upon the numbers of bacteria and fungi in
the soil.

NUMBERS OF MICROORGANISMS 45

Numbers of protozoa in the soil. The protozoa exist in the soil both
in an active stage and in a cyst condition. The methods employed
should give more or less accurate information concerning the numbers
of protozoa in the soil in both of these two stages. It is sometimes
essential to be able to count protozoa in culture solutions. This can
be done in four different ways. (1) The most common method con-
sists in placing a drop of a known volume of the protozoan suspension
under the microscope and counting the number of protozoa. The use
of a slimy colloidal solution for the purpose of reducing the motility of
protozoa, as, for example, semen psylii, semen cydoniae or gum tragacanth
has been suggested. 100 Two per cent gelatin solution may also be
employed for this purpose. (2) The use of a standard loop devised for
bacteria may also be employed 101 - 102 for counting of protozoa. (3) The
agar plate method may be used. 103 None of these three methods is very
suitable for the determination of the number of protozoa in the soil
itself, although the first two can be used for the examination of soil for
the presence of living protozoa. For an accurate determination of the
total number of protozoa in the soil, including both the active forms and
the cysts, the (4) dilution method is most appropriate. 104 ' 105 ' 106 The
method consists in diluting the soil with sterile tap water, then placing
1 cc. portions of the various dilutions into sterile media, incubating,
examining the cultures at periodic intervals (5, 12 and 20 days) and
determining the highest dilution at which growth takes place. The
number of protozoa is thus found to lie between this and the next
higher dilution at which growth did not take place. If, for example,
a dilution of 1:1000 gives growth, while 1:10,000 does not give any,
there are between 1000 and 10,000 protozoa in a gram of soil. The

100 Statkewitsch, P. Zur Methodik der biologischen Untersuchungen liber die
Protisten. Arch. Protistenk. 5: 17-39. 1905.

101 Muller, P. T. Uber eine neue, rasch arbeitende Methode der bakteri-
ologischen Wasseruntersuchung und ihre Anwendung auf die Prufung von Brun-
nen und Filterwerken. Arch. Hyg. 75: 189-223. 1912.

102 Koch, G. P. Soil protozoa. Jour. Agr. Res. 4: 511-559. 1915;5:477-488.
1915; Soil Sci. 2: 163. 1916.

103 Killer, J. Die Zahlung der Protozoen im Boden. Centrbl. Bakt. II, 37:
521-534. 1913.

104 Rahn, O. Methode zur Schatzung der Anzahl von Protozoen im Boden.
Centrbl. Bakt. II, 36: 419-421. 1913.

108 Cunningham, A. Studies on soil protozoa. Jour. Agr. Sci. 7: 49-74. 1915.
106 Sherman, J. M. The number and growth of protozoa in soil. Centrbl.
Bakt. II, 41: 625-630. 1914; also 1916 (p. 47).

46 PRINCIPLES OF SOIL MICROBIOLOGY

dilutions can be made narrower and the number of protozoa determined
with a greater degree of accuracy. This method can also be modified so
as to give not only the total number of protozoa in the soil, but also the
number of active forms and cysts. 107

The soil is passed through a 3 mm. sieve and two 10-gram samples are taken.
The total number of protozoa (active forms plus cysts) is determined, in one sam-
ple, while the other sample is used for the determination of cysts. Taking care to
agitate the flask while the liquid is being transferred, the following dilutions are
prepared:

1

10 grams

of soil

in

100 cc.

H 2

=

1:10

dilution

2

10 cc.

of No.

1 in

90 cc.

H 2

=

1:100

dilution

3

10 cc.

of No.

2 in

90 cc.

H 2

=

1:1000

dilution

4

20 cc.

of No.

3 in

30 cc.

H 2

=

1:2500

dilution

5

20 cc.

of No.

4 in

20 cc.

H 2

=

1:5000

dilution

6

30 cc.

of No.

5 in

15 cc.

H 2

=

1:7500

dilution

7

30 cc.

of No.

6 in

10 cc.

H 2

=

1:10,000

dilution

8

20 cc.

of No.

7 in

30 cc.

H 2

=

1:25,000

dilution

9

20 cc.

of No.

8 in

20 cc.

H 2

=

1:50,000

dilution

10

30 cc.

of No.

9 in

15 cc.

H 2

=

1:75,000

dilution

11

30 cc.

of No.

10 in

10 cc.

H 2

=

1:100,000 dilution

Dilutions of 25-102,400 or 50 204,800 may also be prepared.

Nutrient agar is poured into a series of sterile Petri dishes. When the medium
has solidified, the dishes are inoculated in pairs with 1 cc. of each dilution. The
same pipette may be used when one begins with the highest dilution and goes back
to the lowest. The plates are incubated at 18° to 20° for 28 days and examined
after 7, 14, 21, and 28 days. A little sterile water is added to the plates, in case
they begin to dry out. The examination is made by scraping off a little of the
surface of the plate, placing on a slide under the microscope and examining with
low and high power.

The second 10-gram sample of soil is treated with sufficient 2 per cent HC1,
over night, to neutralize the carbonate present in the soil and still leave an excess
of unchanged 2 per cent HC1. This kills all the active forms leaving the cysts
unharmed. The number of protozoa is then determined by the same dilution
method. The number of cysts subtracted from the total number of organisms,
obtained in the first count, gives the number of active protozoa per gram of soil.

Fisher 108 investigated the theory of estimation of microorganisms by
the dilution method and found that a very high efficiency can be thus
obtained (87.71 per cent). By the use of a special table, the number
of protozoa can be calculated, when the number of negative plates is

107 Cutler, D. W. A method for estimating the number of active protozoa in
the soil. Jour. Agr. Sci. 10: 135-143. 1920.

108 Fischer, R. A. On the mathematical foundations of theoretical statistics.
Phil. Trans. Roy. Soc. London. Ser. A, 222: 309-368. 1922.

NUMBERS OF MICROORGANISMS

47

known. By using 26 plates and a series of dilutions (13) ranging from
ts to TFS.Vinrj the following results were obtained: 109

NUMBER OF STERILE
PLATES

PROTOZOA PER GRAM

NUMBER OF STERILE
PLATES

PROTOZOA PER GRAM

1

110,000

14

640

2

59,000

15

450

3

36,000

16

320

4

23,000

17

230

5

16,000

18

160

6

11,000

19

110

7

7,600

20

79

8

5,300

21

56

9

3,700

22

3S

10

2,600

23

25

11

1,800

24

15

12

1,300

25

6.8

13

900

For the examination of trophic protozoa, the soil sample is moistened
with some sterile water, placed on a slide and examined for a minute
under the microscope.

When soil or soil suspension is placed in a medium favorable for the
development of protozoa, there is found a close connection between the
growth of these organisms to that of the bacteria, the former always
lagging behind. There is also observed a general sequence of protozoan
forms, the flagellates appearing first, followed by the ciliates and later
by the amoebae. A detailed review of the media used for the cultiva-
tion of protozoa is given elsewhere (p. 317).

Garden soils contain a great number of protozoa; 110 amoebae were
found 111 to develop on agar plates, even when the inoculum was only
1 mgm. of soil. An average of about 100 ciliates and 1000 to 10,000
flagellates per gram of soil was reported by earlier workers. 112, m
Sherman 113 compared the numbers of protozoa in sixteen soils represent-
ing various soil types under various treatments; he found that normal

109 Cutler etal., 1922 (p. 32).

110 Hiltner, 1907 (p. 47).

111 Stormer, K. Uber die Wirkung des Schweffelkohlenstoffs und ahnlicher
Stoffe auf den Boden. Jahresb. Ver. angew. Bot. for 1907, 5: 113-131. 1908.

112 Rahn, 1913 (p. 45).

113 Sherman, J. M. Studies on soil protozoa and their relation to the bacterial
flora. Jour. Bact. 1: 35-66; 165-185. 1916.

U4 Waksman, S. A. Studies on soil protozoa. Soil Sci. 1: 135-152; 2: 363-
376. 1916.

48

PRINCIPLES OF SOIL MICROBIOLOGY

fertile soil has a protozoan content approximating 10,000 per gram, the
flagellates constituting the greater portion; about 100 ciliates were
found per gram; certain types of protozoa are active in the soil under

TABLE 9
Influence of organic matter on numbers of protozoa in the soil {average of 10 counts) 117

BROADBALK MANURED

(10 PER CENT ORGANIC

MATTER)

harpenden field

(5.7 per cent organic

matter)

C*

20
130
120

20

F

A

C

F

A

February 1-March 5, 1917

32,000
23,000
31,200
42,300

1500

1600

18,600

23,200

10
20
10
10

13,500
71,000
25,700
23,330

500

April 17- June 6, 1917

500

June 13-September 20, 1917

October 10-December 15, 1917

17,000
10,000

* C = ciliates, F = flagellates, A = amoebae.

TABLE 10
Influence of soil depth upon the numbers of protozoa 111

DEPTH

AMOEBAE PER 1 GRAM

FLAGELLATES PER 1
GRAM

CILIATES PER 1 GRAM

inches

6
12

18

1750

8750
100
100

100

TABLE 11
Numbers of protozoa and bacteria per gram of soil from two Broadbalk plots 119

MANURED

UNMANURED

ACTIVE
FORMS

APPROXI-
MATE

Winter total

Summer
total

Winter
total

Summer
total

DIAMETER

Flagellates

Amoebae

150,000
5,000

600,000

15,000

1,000

200

24,000,000

5,500
40

15,000
2,000

15 to 95
per
cent
in all
cases

7.5-15
8-22

Thecomoebae . .

15

50
10,000,000

20-40

Bacteria

4,000,000

5,000,000

1-4

normal and even sub-normal conditions of moisture, the active forms
being probably restricted to the flagellates. Although the amoebae are
also widely distributed in the soil, 115 their numbers have not been re-

116 Martin, C. H., and Lewin, K. R. Some notes on soil protozoa. Phil.
Trans. Roy. Soc. London, 205B: 77-94. 1914; Jour. Agr. Sci. 7: 106-119. 1915.

NUMBERS OF MICROORGANISMS

49

ported in detail due to the difficulty of their development on the com-
mon media. In soils with a moisture content not in excess of the
physical optimum, protozoa exist mainly in a nontrophic state. 116 The
protozoa become active in the soil whenever there is excessive moisture
present for a period of several hours. Such conditions are common,
especially in rainy seasons, in poorly drained soils, in spring, etc. The
protozoa then excyst and, after a period of active growth, they repro-
duce. When conditions become again unfavorable, they either encyst
or die. Protozoa are readily found in field ditches, furrows with stand-
ing water, etc. Since the protozoa chiefly use bacteria as their food

40.000

Jfl

jj 35.000

^

S 30.000

■fry

%

c

i. 5

%

—

» '

\»

. ;rl

fj

Cr -1

-._

1 —

-—

3

:-. £

\WSffl

C/ 00

$ fc 15.000

i " '

0..

...c

\

\ °

■O"

o

\

o

\

E

J^--°

\P

0*

\l\

o ...

,-v

20
18 I
16 |.25
14 \. ■<&
12 |20|

10 I I
8 Sl5*

el

"9

8 15 22 29
October

5 12 19 26
Nnvfml)or

3 10 17 24 31 7
December

14 21 28

Jancary

Fig. 6. Numbers of active flagellates, amoebae and bacteria in the soil, in
relation to its moisture content (from Cutler and Crump).

(perhaps also organic matter and soil extracts, especially in the case of
small flagellates), they are more abundant in rich fertile soils than in poor
acid soils. Their greatest numbers are concentrated in the top four or
six inches of soil, where the bacteria are also at a maximum, while below
twelve inches the soil is practically free from protozoa. 117 Isolated
species may be found, usually in a cyst condition, even at very low
depths. 118 By the use of the method above described, Cutler and

116 Fellers, C. R., and Allison, F. E. The protozoan fauna of the soils of New
Jersey. SoilSci.9: 1-25. 1920.

117 Crump, L. M. Numbers of protozoa in certain Rothamsted soils. Jour.
Agr. Sci. 10: 182-198. 1920.

118 Sandon, 1927 (p. 329).

50

PRINCIPLES OF SOIL MICROBIOLOGY

Crump 119 estimated at frequent intervals the numbers of active pro-
tozoa and cysts in Rothamsted soils and found them to be much higher
on the manured than on the unmanured plot.

The numbers of protozoa and bacteria were found to vary from day
to day. 120 An inverse relation was found between the number of active
amoebae and bacteria.

The numbers of algae in the soil can also be determined by the dilu-
tion method, using the specific media, described elsewhere (p. 221) best

Days 1

Fig. 7. Daily variation of active flagellates in the soil (from Cutler and
Crump).

adapted to the development of these organisms. 121 The numbers of
nematodes and other worms, insects and other members of the inverte-
brate population of the soil, as well as methods of determination, are
given elsewhere (p. 342).

119 Cutler, D. W., and Crump, L. M. Daily periodicity in the numbers of active
soil flagellates : with a brief note on the relation of trophic amoebae and bacterial
numbers. Ann. Appl. Biol. 7: 11-24. 1920.

120 Cutler et al., 1922 (p. 32).
111 Bristol-Roach, 1926 (p 225V

PART B

ISOLATION, IDENTIFICATION,

AND CULTIVATION

OF SOIL MICROORGANISMS

CHAPTER II

Pure Culture Study and Classification of Soil Bacteria

It is almost impossible at present to make a complete study of the
various types of bacteria occurring in the soil, due both to the great
variety of forms and to the lack of sufficient knowledge concerning
many of them. Bacteria offer but few stable characteristics which can
be utilized for their separation. Classification of bacteria in general
and of soil bacteria, in particular, is not fully satisfactory. The different
systems of classification can, therefore, be merely tentative; they are
based either on the physiological activities of the organisms, especially
their carbon, nitrogen, and oxygen metabolism, or on their morphological
relationships. In the general subdivision of the soil bacteria, the physio-
logical system will be utilized for the greater convenience which it
offers in the study of the role of these organisms in the soil; in the
differentiation of the individual bacteria within the large groups morpho-
logical characters are utilized.

Pure culture study. In the case of large organisms, a pure culture
corresponds to an individual. In the case of the unicellular bacteria,
the transformation carried on by one cell is too small, and large num-
bers of organisms must take part in a reaction, before measurable
changes are obtained; these individual cells, however, must be as much
alike as possible. The various bacteria do not exist in the soil in pure
culture, but in mixed associations; entirely different results are usually
obtained under natural conditions, with the various microbiological
processes completing one another, than in pure culture. This is
especially true in the case of organisms which depend for their nutrients
upon the activities of other organisms, as in the case of the nitrite
bacteria which depend upon the various proteolytic organisms for
their substrate (ammonia), and the nitrate-forming bacteria which
depend upon the nitrite formers for their supply of energy source
(nitrite). Antagonistic effects are often marked under natural sur-
roundings, while they are eliminated in pure culture. Certain soil
processes, such as cellulose decomposition or nitrogen-fixation, are
carried on much more actively by crude than by pure cultures of the
organisms concerned, due to the fact that the accompanying organisms

53

54 PRINCIPLES OF SOIL MICROBIOLOGY

either use up the deleterious waste products or in some other way-
contribute to the process in question.

To be able to identify the various species of bacteria and determine
their specific functions upon nutritive media and in the soil, it is neces-
sary to obtain them in pure culture. A mere microscopic examination
of bacteria is usually insufficient for their identification. They should
be studied on artificial culture media and records made of the cultural
characteristics and biochemical changes produced. Among these
characteristics, we may include size, appearance and color of colony;
growth upon solid and liquid media; modification of color, consistency,
reaction and chemical composition of the medium; action upon sugars
and proteins and enzyme formation, and influence of oxygen tension,
temperature and chemical agents. Among the morphological charac-
teristics, we may include form, size, grouping and appearance of cells,
motility, spore formation, manner of reproduction, etc.

Pure cultures of bacteria and other microorganisms are often weak-
ened, when grown on artificial culture media, or may lose altogether
their capacity of producing the change which they carry on in nature,
as in the case of certain nitrogen-fixing bacteria. This leads to differ-
ences in results in the hands of different investigators. We are dealing
here with organisms whose functions are changeable and which are very
variable in their activities. 1 It is best to work with freshly isolated
organisms and it is always advisable to establish the constancy of
physiological characteristics.

In obtaining pure cultures of a number of soil bacteria, especially
those producing certain biochemical changes when grown in the presence
of specific substances, the selective or accumulative culture method used
extensively by Beijerinck, 2 Winogradsky and others may be utilized.
The first step made in the isolation of a particular organism consists in
adding some of the material (soil or manure) to a liquid medium con-
taining nutrients especially adapted to the growth of that particular
organism. This allows the accumulation of the organism in question
and eliminates largely the other accompanying forms. By transferring
repeatedly to the same sterile medium, an enrichment culture is ob-
tained. Theoretically one species is obtained under ideal enrichment

1 Pringsheim, H. Die Variabilitiit niederer Organismen. Berlin. J.
Springer. 1910.

2 Richter, O. Die Bedeutung der Reinkultur. Borntraeger. Berlin. 1907;
Stockhausen. Oekologie, Anhaufungen nach Beijerinck. 1907.

PURE CULTURE STUDY OF SOIL BACTERIA 55

conditions; actually, a number of strains may be obtained, which, on
separation, may often be mistaken for different species.

The direct method of isolation suggested by Winogradsky 3 may also
be employed. This consists in adding a specific nutrient to a silica gel
plate, containing the necessary nutrients, and inoculating with particles
of soil. Only the organisms capable of acting upon the specific substrate
will develop on the plate in forms readily isolated.

In the case of the majority of soil bacteria (especially the
heterotrophic forms), both aerobic or anaerobic, the plate method
will prove convenient for the isolation of the individual organisms
from colonies. The selective culture method in most cases and
the plate method in some cases yield only crude cultures of the
organisms. In the majority of cases these cultures are quite satisfac-
tory. 4 However, for biochemical studies and especially in the study
of life cycles of bacteria, it is advisable to obtain single-cell cultures of
the organisms. In most instances this is accomplished by the dilution
method, which may be accompanied by the transfer of a single cell by
the India ink 5 method or by means of a capillary pipette. 6 The first
consists in mixing a highly diluted culture with sterile India or China
ink, placing pin-point droplets of the mixture upon the surface of sterile
agar or gelatin, covering with sterile cover slips, examining with high-
power objective for the presence of a single cell, incubating, and finally
transferring or lifting the cover slip with adhering bacterial cell and
inoculating into sterile medium. The second method consists in

3 Winogradsky, 1925 (p. 11).

4 Lohnis, F. Studies upon the life cycles of the bacteria. I. Mem. Nat. Acad.
Sci. 16, 1921, No. 2, p. 39.

6 Burri, R. Eine einfache Methode zur Reinziichtung von Bakterien unter
mikroskopischer Kontrolle des Ausgangs von der einzelnen Zelle. Centrbl. Bakt.
II, 20: 95-96. 1908; Das Tuschepunktverfahren. G. Fischer. Jena. 1909.

6 Barber, M. A. On heredity in certain micro-organisms. Kansas Univ
Science Bull. 4: no. 1 48 p. 1907; Jour. Inf. Dis. 5: 379. 1908; 8: 348. 1911
The pipette method in the isolation of single microorganisms and in the inocula
tion of substances into living cells. Philippine Jour. Sci. B. 9: 307-360. 1914
The use of single cells method in obtaining pure cultures of anaerobes. Jour
Exp. Med. 32: 295. 1920; Shouten, S. L. Reinkulturen aus einer unter dem
Mikroskop isolierten Zelle. Ztschr. Wissensch. Mikrosk. 22: 10, 1907; 24: 258
1907; Hecker, F. A new model of double pipette holder and the technic for the
isolation of living organisms. Jour. Inf. Dis. 19: 305. 1916; Hort. Jour. Hyg
18: 361. 1920; Topley, W. W. C, Barnard, J. E., and Wilson, G. S. A new
method of obtaining cultures from single bacterial cells. Jour. Hyg. 20 : 221-226
1921; Malone, R. H., A simple apparatus for isolating single microorganisms
Jour. Path. Bact. 22: 222. 1918.

56 PRINCIPLES OF SOIL MICROBIOLOGY

drawing up individual cells by means of the Barber capillary pipette
and transferring them to specific sterile liquid media. The micro-
manipulator of Chambers 7 may also be employed.

In view of the fact that the bacteria live and act in the soil in mixed
culture, important information is obtained not only from the study of
pure cultures, but also from mixed cultures, either crude or artificially
prepared.

For a description of pure cultures of bacteria, the Chart of the Society
of American Bacteriologists can be used . 8 • 9 This includes a study of the
important morphological, physiological and cultural characteristics
of the organisms. However, the chart can not be readily utilized in
the description of most of the soil bacteria with the exception of some
of the heterotrophic bacteria requiring combined nitrogen, especially
the spore-forming organisms. 10

Differentiating characters of bacteria. The size of the organisms is a
variable factor with rather large limits of variation which depend upon
the nutrient media in which the cells are grown and various environ-
mental conditions. 11 However, in the case of the spore-forming bacteria,
the size of the spore is of great diagnostic value. Motility of bacteria
has been utilized in the classification of Migula but the constancy and
value of this character have been questioned by Lehmann and Neu-
mann, 12 who found the presence or absence of spore production by

7 Chambers, R. New micromanipulator and methods for the isolation of a
single bacterium and the manipulation of living cells. Jour. Inf. Dis. 31 : 334-343,
344-348. 1922.

8 Harding, H. A. The constancy of certain physiological characteristics in the
classification of bacteria. N. Y. Agr. Exp. Sta. Tech. Bui. 13. 1910.

9 Rahn, O., and Harding, H. A. Die Bemi'ihungen zur einheitlichen Beschrie-
bung der Bakterien in America. Centrbl. Bakt. II, 42 : 385-393. 1914.

10 The methods used in the description of these organisms are discussed in the
various Reports of the Committee on Bacteriological Technic of the Society of
American Bacteriologists. C. B. T. Methods of pure culture study. Jour.
Bact. 3: 115-138. 1918; 4: 107-132. 1919; 5: 127-143. 1920; 7: 107-132. 1919.
Conn, H. J. et al. Recent work on the descriptive chart and the manual of
methods. Ibid. 10: 315-319. 1925. Also Conn, H. J. Soil flora studies. II.
Methods best adapted to the study of the soil bacteria. N. Y. Agr. Exp. Sta.
Tech. Bui. 57: 18-42. 1917.

11 Lohnis, F., and Smith, N. R. Life cycles of bacteria. Jour. Agr. Res.
6: 676-702. 1916; 23: 401-432. 1923; also Lohnis, 1921 (p. 55); Scales, F. M.
Induced morphologic variation in B. coli. Jour. Inf. Dis. 29: 591-610. 1921.

12 Lehmann, K. B., and Neumann, R. O. Atlas und Grundrisz der Bakterio-
logie. Mi'inchen, Lehmann's. 1920.

PURE CULTURE STUDY OF SOIL BACTERIA 57

bacteria to be a more definite characteristic. The form of growth of the
various organisms is also of important diagnostic value.

As to the various physiological characteristics, some, like liquefaction
of gelation, nitrate reduction 13 and chromogenesis, are of considerable
diagnostic value; others, like diastatic power, fermentation of sugars,
indol formation, action on milk, give rather inconsistant results.

Although the morphological characters of the bacteria should be uti-
lized in a scientific system of classification, the soil bacteria lend them-
selves readily to a general classification based upon their physiological
activities. Such a system is suggested here for the purpose of grouping
the bacteria according to the important soil processes in which they
take an active part, utilizing the more systematic classification for the
discussion of the specific organisms.

Life cycles of bacteria. According to Lohnis, bacteria live alternately
in an organized and in an amorphous or "symplastic" stage. In the
latter stage the living matter which has been previously enclosed in the
separate cell, undergoes a thorough mixing either by a complete dis-
integration of the cell wall and cell contents or by a "melting together"
of the contents of many cells which leave their empty cell walls behind
them; it seems to be formed both of the vegetative and reproductive
cells; this process is similar to autolysis, without the destruction of the
living substance. The symplasm may undergo amoeboid changes or
become encapsulated, giving spherical macrocysts. In the process of
formation of new individual cells from the symplasm, "regenerative
units" are first visible, which increase in size becoming either directly
vegetative cells or "regenerative bodies." The latter become, by
germination and stretching, normal cells or return temporarily into the
symplastic stage.

In addition to symplasm formation, two or more individual cells may
unite directly (conjunction). All bacteria multiply not only by fission
but also by the formation of gonidia, which first become regenerative
bodies or exospores. The gonidia may either grow directly into full
cells or enter the symplastic stage. Thread-like branching forms may
also be produced from the symplasm. The life cycles of each species
of bacteria is composed of several sub-cycles showing wide morphologi-
cal and physiological differences. They are connected with each other
by the symplastic stage (Nos. 47, 48, PL IX) . However, some investi-

" Breed, R. S. The standard method of determining nitrate reduction.
Science, N. S. 41: 661. 1914.

58 PRINCIPLES OF SOIL MICROBIOLOGY

gators 14 deny the formation of symplasm by Azotobacter; this is
looked upon as a stage of gradual autolysis of the cell membrane
rather than a stage of reproduction. Henrici 15 demonstrated that bac-
terial cells undergo metamorphosis during the growth of a culture,
similar to that exhibited by cells of a multicellular organism; each
species presents three types of cells: a young form, an adult form and
a senescent form; the variations depend upon the rate of metabolism.
Classification of bacteria. This is not the place to discuss the value
of the different systems of classification of bacteria. It is sufficient to
call attention to the systems of Migula, Lehmann and Neumann and
to that proposed by the Committee on Classification of the Society
of American Bacteriologists, and incorporated in Bergey's Manual. 16
One may conveniently use here Migula's classification, with slight modi-
fications, especially in the case of the Bacteriaceae, which are more
commonly divided on the basis of spore formation.

I. Simple and undifferentiated forms, not forming any threads and not branch-
ing under normal conditions. Order Eubacteria.

1. Cells mostly spherical, rarely rod-shaped, Coccaceae Zopf emend.

Migula:

(a) Division in one direction, frequent formation of chains, 1. Strep-

tococcus Billroth.

(b) Irregular division in all directions; cells occur singly, in pairs or

clumps, but not in chains, 2. Micrococcus Cohn.

(c) Division in three directions leading to packet formation, 3.

Sarcina Goodsir.
Planostreptococcus, Planococcus, Planosarcina include similar
but flagellated group.

2. Cells mostly rod-shaped, rarely spherical or curved, Bacteriaceae Zopf

emend. Migula:

(a) No endospores formed, 4. Bacterium Cohn.

(b) Endospores formed, 5. Bacillus Cohn.

(c) Cells with polar flagella; endospores rarely formed, 6. Pseiulo-

monas.

3. Cells mostly curved or spiral, rarely spherical or rod-shaped, Spirilea-

ceae Migula:

(a) Cells comma-shaped, 7. Vibrio Midler emend. Loftier.

(b) Cells rigid, spiral shaped, 8. Spirillum Ehrenberg emend Loffler.

(c) Cells flexible, spiral shaped, 9. Spirochaeta Ehrenberg.

14 Beauverie, J. Le symplasme bacterien existe-t-il? Cas de l'azotobacter.
Compt. Rend. Acad. Sci. 180: 1792-1794. 1925.

15 Henrici, A. T. On cytomorphosis in bacteria. Science 61: 644 646. 1925.

16 Winslow, C.-E. A., Broadhurst, J., Buchanan, R. E., Krumwiede, C, Rogers,
L. A., and Smith, G. H. The families and genera of the bacteria. Jour. Bact.
6: 191-229. 1920; also Ibid. 2: 505. 1917; Bergey, 1924 (p. x).

PURE CULTURE STUDY OF SOIL BACTERIA 59

II. Growth similar to the Eubacteria, but cells containing bacteriopurpurin
(and bacteriochlorin), being colored rose, red or violet. Order Rhodo-

BACTERIA.

III. Colorless bacteria accumulating sulfur within their cells. Order Thio-

BACTERIA.

IV. Alga-like bacteria. Order Phycobacteria. This order comprises the

family Chlamydobacteriaceae which form sheaths; these include the

iron bacteria. 17
V. Fungus-like bacteria, rod-shaped, rarely filamentous. Order Mycobacteria.

The Actinomyces are frequently included in this genus.
VI. Bacterial cells enclosed in a slimy mass, forming a pseudoplasmodium-like

aggregation before passing into a cyst-producing resting stage. Order

Myxobacteria.

Classification of soil bacteria based upon their physiological activities 1 *

I. Autotrophic and facultative autotrophic bacteria, deriving their carbon
primarily from the C0 2 of the atmosphere and their energy from the
oxidation of inorganic substances or simple compounds of carbon.

1. Bacteria using nitrogen compounds as sources of energy:

(a) Nitrite forming bacteria.

(b) Nitrate forming bacteria.

2. Bacteria using sulfur and sulfur compounds as sources of energy.

3. Bacteria using iron (and manganese) compounds as sources of energy.

4. Bacteria using simple carbon compounds as sources of energy:

(a) Bacteria oxidizing carbon monoxide.

(b) Bacteria oxidizing methane.

5. Bacteria using hydrogen as a source of energy.

II. Heterotrophic bacteria deriving their carbon and energy from various
organic compounds:
1. Nitrogen-fixing bacteria, deriving their nitrogen from the atmosphere,
in the form of gaseous atmospheric nitrogen:

a. Non-symbiotic nitrogen-fixing bacteria:

(a) Anaerobic types: Butyric acid bacteria {Bac. amylobacler) ,

including species of Clostridium, Granulobacter, etc.

(b) Aerobic types: Azotobacter, Radiobacter, Bad. aerogenes,

Bad. pneumoniae, etc.

b. Symbiotic nitrogen-fixing (nodule) bacteria.

17 A detailed review of "The generic names of bacteria" is given by E. M. A.
Enlows. Hyg. Lab. Bui. 121. Washington, D. C, 1920; Buchanan, R. E.
Systematic bacteriology. 1925.

18 A detailed system of bacteria based on biochemical relationships has been
proposed by Orla-Jensen— Die Hauptlinien des natiirlichen Bakteriensystems.
Centrbl. Eakt. II, 22: 305 346. 1909.

60 PRINCIPLES OF SOIL MICROBIOLOGY

2. Aerobic bacteria requiring combined nitrogen:

a. Spore producing bacteria.

b. Non-spore producing bacteria.

3. Anaerobic bacteria, requiring combined nitrogen.

For the sake of convenience three other groups of bacteria may be discussed
separately, due to their specific physiological activities and importance in
certain soil processes; these bacteria belong, according to their morphology and
general physiology, to groups 2 and 3, namely :

4. Cellulose decomposing bacteria.

5. Urea and uric acid bacteria.

6. Denitrifying bacteria.

S. WlXOGRADSKT

CHAPTER III

Autotrophic Bacteria

The nature of autotrophic bacteria. Autotrophic bacteria are organisms
which are capable of obtaining their carbon from C0 2 of the atmosphere
and their energy from the oxidation of inorganic substances, including
simple inorganic compounds (or the elementary form) of nitrogen,
sulfur, iron, hydrogen and carbon. Some of these transformations,
particularly those of the nitrogen and the sulfur compounds are of great
importance in the soil.

Obligate autotrophic bacteria, or anorgoxydants, are characterized 1
by a series of physiological properties, which differentiate them sharply
from the rest of the bacteria. These properties are as follows:

1. Their development in nature takes place only in strongly elective,
almost pure mineral media, which contain specific oxidizable inorganic
substances.

2. Their existence is connected with the presence of these substances,
which undergo oxidation as a result of the life activities of the organisms.

3. This oxidation process supplies their only source of energy.

4. The organisms do not need any organic nutrients for structure or
for energy.

5. They are almost incapable of decomposing organic matter and may
even be checked in their development by its presence.

6. They use, as an exclusive source of carbon, carbon dioxide, which is
assimilated chemosynthetically.

These original conceptions of Winogradsky may be modified at
present in two respects:

1. The presence of organic matter may not prove injurious to the
activities of the autotrophic bacteria. As a matter of fact, the presence
of small quantities of certain organic substances may even be stimulating
to some. Further, the existence of these organisms in the soil, where
they carry on their life processes, takes place in the presence of soluble
organic substances.

1 Winogradsky, S. Eisenbakterien als Anorgoxydanten. Centrbl. Bakt. II,
67: 1-21. 1922.

61

62 PRINCIPLES OF SOIL MICROBIOLOGY

2. Only few of the autotrophic bacteria are obligate, some are faculta-
tive autotrophic. The latter, as in the case of some sulfur, hydrogen
and methane bacteria, can exist both autotrophically and hetero-
trophically.

Bacteria deriving their energy from nitrogen compounds. The bacteria
which are able to derive their energy from the oxidation of simple forms
of nitrogen (nitrifying bacteria) are divided into two groups; (1) those
that oxidize ammonium salts to nitrites, and (2) those that oxidize
nitrites to nitrates. The latter comprise rather limited groups of micro-
organisms, both in their morphology and physiology. Representatives
of both groups were isolated and described by Winogradsky 2 in 1891 and
1892.

Various purely chemical theories were suggested at different times to
explain the process of nitrification in nature. Pasteur 3 was the first
to suggest that the oxidation of ammonia to nitrate is accomplished
by the agency of microorganisms. This view was definitely confirmed
(1877) by Schlosing and Mi'mtz, 4 who demonstrated that the heating of
soil, otherwise capable of rapidly transforming ammonia to nitrites, to
100°C. or treating it with antiseptics (chloroform) was sufficient to pre-
vent nitrification; when afresh portion of soil was added to the treated
soil, the power of transforming ammonia to nitrates was restored. Aera-
tion was found to be essential to nitrification. It was obtained either
by bubbling air through the medium, or by spreading the medium in a
thin layer over the bottom of the container. According to Schlosing, 5
oxygen is taken up during the process of nitrification and the quantity of
oxygen consumed bears a constant ratio to the amount of nitrogen nitri-
fied. A temperature of about 37°C. and the presence of calcium carbonate
or alkaline carbonates in low concentrations (0.2 to 0.5 per cent) were
also found to be favorable. These investigations proved that the condi-
tions commonly utilized in the saltpeter heaps were quite essential for the
activities of the organisms; namely, (1) the presence of nitrogenous

2 Winogradsky, S. Recherches sur les organismes de la nitrification. Contri-
butions a la morphologie des organismes de la nitrification. Arch. Sci. Biol.,
St. Petersbourg, 1: 87, 127. 1892.

3 Pasteur, L. Etudes sur les mycodermes. Role de ces plantes dans la fer-
mentation acetique. Compt. Rend. Acad. Sci. 54: 265-270. 1862.

4 Schlosing, Th. and Miintz, A. Sur la nitrification par les ferments organises.
Compt. Rend. Acad. Sci. 84: 401. 1877; 85: 1018-1020. 1877; 86: 892. 1878;
89: 891-4, 1047-7. 1879.

* Schlosing, Th. Sur la nitrification de l'ammoniaque. Compt. Rend. Acad.
Sci. 109: 423^28. 1889.

AUTOTROPHIC BACTERIA 63

organic compounds thoroughly mixed with the soil, (2) a thorough aera-
tion of all the layers of the heap, (3) a proper moisture content kept up
by moistening the heap at regular intervals, (4) the presence of bases,
like calcium carbonate (or soap water). The activities were found to be
noticeable at 5°C, became prominent at 12° and reached a maximum
at 37°. Higher temperatures (45°) exerted an injurious effect and at
55°C the process came to a standstill.

Warington 6 confirmed the biological nature of nitrification not only
in soil, but also in ammoniacal solutions inoculated with soil. He
studied (1) the influence of organic substances upon nitrification, (2)
the abundant production of nitrites in the process of nitrification and (3)
the nitrification of organic nitrogen. Warington observed that in a
liquid medium containing ammonium chloride, chalk, and sodium-
potassium tartrate, the ammonia could be acted upon only to a very
limited extent; when sugar replaced the tartrate, there was a decided
injurious effect upon the process of nitrification, which could not be
explained, since the nature of the organism was unknown. The relative
amounts of nitrite and nitrate formed in the process of nitrification
presented another unexplainable difficulty. As to the nitrification of
organic nitrogenous substances (urine, milk, asparagine.) , Warington
demonstrated that this has to be preceded first by their transformation
into ammonia; in other words, only those substances can be nitrified
which can first be converted into ammonia. It was soon found that
nitrites arose from the oxidation of ammonia and not from the reduction
of nitrates; Munro 7 distinguished ammonia formation from ammonia
oxidation (nitrification) and, without suspecting the existence of the
different organisms, he had a rather clear idea of the two processes.

All attempts to isolate the specific organisms concerned in the process
of nitrification failed, in spite of the fact that nitrification is an important
biological process not only in the soil but also in sewage purification.
This was chiefly due to the fact that the proper methods were lacking.
Although claims have been put forth by various investigators 8 that a

6 Warington, R. On nitrification. Jour. Chem. Soc. (London), 33: 44-51.
1878;35:429-456. 1879;45:637-672. 1884; Chem. News. 61: 135. 1890; Trans.
Chem. Soc. London, 59: 484-529. 1891.

7 Munro, J. H. M. The formation and destruction of nitrates and nitrites in
artificial solutions and in river and well water. Jour. Chem. Soc. 49: 632-681.
1886.

8 Heraeus, W. Uber das Verhalten der Bakterien im Brunnenwasser, sowie
uber reducierende und oxydierende Eigenschaften der Bakterien. Ztschr. Hyg.
1: 193-235. 1886.

64 PRINCIPLES OF SOIL MICROBIOLOGY

number of organisms, some even pathogenic in nature, are able to pro-
duce nitrates, they were valueless due to improper interpretation. The
traces of nitrates probably came from the atmosphere and the nitrites
from the reduction of the nitrates present in the medium. 9 The
French 4 - 5 and English 6 - 10 investigators were primarily chemists, while
the German bacteriologists 8 were so much under the influence of the
gelatin plate method of R. Koch that the absence of growth on that
medium was thought to indicate the entire absence of an organism.

Winogradsky started out with the idea that we were dealing here with
an organism of unknown properties which does not develop on the
gelatin plate. Fresh from his work on the sulfur and iron bacteria
(1885-1888), whereby he recognized organisms which can derive their
energy from inorganic compounds, he reasoned that a source of energy
so abundant as ammonia would be likely to be utilized by microorgan-
isms. If so, the organisms concerned might show properties similar to
those of organisms oxidizing other inorganic substances. The principle of
elective culture was adopted, whereby conditions are made unfavorable
for the development of any other organism, except those that are able
to oxidize ammonium compounds. Winogradsky used a medium of the
following compositions:

Ammonium sulfate 1 gram

Potassium phosphate. 1 gram

Tap water 1000 cc.

Portions of this medium (100 cc.) were placed in flasks each of which
contained 0.5 to 1 gram basic magnesium carbonate. The flasks were
inoculated with a little soil and, when active nitrification took place,
transfers were made to fresh quantities of medium. The ammonia
disappeared in two weeks, while the di-phenylamine reaction for nitrates
appeared in four days and reached its maximum in another three to
four days. Gelatin plates made from the flasks gave various species
of bacteria and yeasts, but none of them was able to nitrify. This
confirmed the idea of Winogradsky that the organism in question
cannot develop on gelatin. After several transfers, the observation
was made that a bacterium was present on the magnesium carbonate
sediment, covering it in the form of a zooglea. Under the microscope,

9 Winogradsky, S. Die Nitrifikation. Lafar's Handb. Tech. Myk. 3: 132
181. 1904.

10 Frankland, G. C, and P. F. The nitrifying process and its specific ferment.
Trans. Roy. Soc. London, B, 181: 107-128. 1890.

AUTOTROPHIC BACTERIA 65

the bacterium from the sediment was found to be regularly oval or
ellipsoidal in nature; it was also present in the medium. In order to
obtain pure cultures of these organisms Winogradsky 11 made use of the
two facts that the bacterium did not grow on the gelatin plate and in
nutrient bouillon but was found abundantly on the magnesium car-
bonate sediment. Some of that sediment was streaked out over a gela-
tin plate and those particles, which remained sterile on the plate, after
a few days incubation, were transferred into fresh flasks containing the
ammonium medium. The final steps in the study and isolation of the
bacteria concerned in the process of nitrification were the separation

Fig. 8. Winogradsky flask for experiments on nitrification. Layer of MgCOj
on bottom of flask and mineral salt solution above it (after Omeliansky).

of the nitrite and nitrate organisms and their cultivation upon specific
media.

For the growth of the nitrite and nitrate forming bacteria, a
thorough aeration of the culture is essential. Winogradsky used
flasks of large diameter (12 cm) with flat bottoms, in which the
liquid formed only a shallow layer (less than 1 cm. in height). Boul-
anger and Massol 12 used scoria and allowed the liquid to reach only
half the height of the scoria layer, which was moistened at regular

11 Winogradsky, S. Recherches sur les organismes de la nitrification. Ann.
Inst. Past. 4: 213-231, 257-274. 1890; 5: 92-100. 1891.

12 Boulanger, E., and Massol, L. Etudes sur les microbes nitrificateurs. Ann.
Inst. Past. 17: 492-515. 1903; 18: 181-196. 1904. Compt. Rend. Acad. Sci.
687. 1905.

66

PRINCIPLES OF SOIL MICROBIOLOGY

intervals, by the shaking of the flask; this hastened the process of nitri-
fication. Still more intensive nitrification was obtained by allowing
the ammoniacal solutions to flow through peat and carbon black inocu-
lated with nitrifying organisms. 13 Ignited soil placed in flat bottomed
Fernbach flasks, combined with a slow rotary movement of the culture
gave intensive nitrification. 14

An alkaline reaction is essential for nitrite and nitrate formation. In
the absence of basic carbonates, only ammonium carbonate is oxidized ;
in the presence of sodium, magnesium or calcium carbonate, free ammonia
as well as the sulfate, phosphate and chloride of ammonium can nitrify. 15
This is due to the fact that the optimum reaction for the organisms is on
the alkaline side of neutrality (pH 7.0 to 8.0). The presence of the base
in the medium is essential as a neutralizing agent to prevent the reac-
tion of the medium from becoming too acid, due to the formation of
nitric and sulfuric acids from the oxidation of the ammonium salt.

In addition to the medium given above, two other media were used: 15,18

(NHO2SO4

K 2 HP0 4

MgS(V7H 2

CaCl 2

NaCl

FeS0 4

Distilled water. ,

Magnesium carbonate (MgC0 3

MEDIUM 2

2 . 0-2 . 5 grams

1.0 grams

. 5 gram

Trace

1000 cc.
Excess (0.5 gram
per flask)

2.0 grams
1 .0 grams
0.5 gram

2.0 grams

0.4 gram

1000 cc.

Excess (0.5 gram

per flask)

The medium is distributed into flasks (about 50 cc. portions), which are then
plugged with cotton and sterilized at 15 pounds pressure for 15 minutes. It is
best to sterilize the ammonium sulfate separately, as a 5 or 10 per cent solution,
and then add it by means of a sterile pipette to the sterile flasks. Gibbs 17 em
ployed medium 3, but reduced the ammonium salt to one gram and substituted
a trace of ferric sulfate for the ferrous salt.

13 Mi\ntz, A., and Laine, E. Utilisation des tourbiers dans la production
intensive des nitrates. Compt. Rend. Acad. Sci. 142: 1239-1244. 1906; also
Ann. Sci. Agron. Ser. 3, 2: 287-395. 1906.

14 Bonazzi, A. On nitrification. Jour. Bact. 4: 43-59. 1919.

15 Winogradsky, 1904 (p. 64).

18 Omeliansky, V. Uber die Isolierung der Nitrifikationsmikroben aus dern
Erdboden. Centrbl. Bakt. II, 5: 537-549. 1899.

17 Gibbs, M. W. The isolation and study of nitrifying bacteria. Soil Science,
8: 427-481. 1919.

AUTOTROPHIC BACTERIA 67

When 1 gram of soil is inoculated into the flasks containing the
culture medium, growth will usually take place at 25° to 30°C after 4
to 5 days, but sometimes only after weeks. Some soils, however, such
as acid peat and certain acid forest soils, 18 may not contain the organisms
in question. They are not very sensitive to drying, 17 but the action
of steam or volatile antiseptics is injurious and results in their destruc-
tion in the soil. Manured and cultivated soils contain the nitrifying
organisms in greatest abundance, especially in the upper layers of soil. 19

The appearance of growth is accompanied by the formation of nitrous
acid and disappearance of ammonia. The former is demonstrated by
the starch-zinc iodide reagent and the latter by Nessler's reagent.
When all the ammonium is oxidized, a fresh portion of ammonium sul-
fate is added; a sterile 10 per cent solution of the salt is kept in a flask
with a plugged graduated pipette: 1 cc. will be equivalent to addition
of 0.2 per cent of the salt to the medium. W T hen a second portion of the
ammonium salt is added, it is oxidized much more rapidly since the
specific organisms have already developed abundantly. A third por-
tion is oxidized even more rapidly, until a certain limit of reaction is
attained depending on the solution of the MgC0 3 and the accumulation
of nitrous acid. The culture is then transferred to a fresh flask with
medium, using preferably a few drops from the bottom of the flask,
since the bacteria form a sediment on the MgC0 3 . Oxidation sets in
now more rapidly and goes on more regularly. After four or five
more transfers and repeated addition of ammonium sulfate to each
culture before a new transfer is made, the culture is rich enough in
the specific organisms and can be used for isolation purposes.

The oxidation of nitrous to nitric acid by the nitrate-forming bac-
teria does not begin until the oxidation of the ammonium salt to nitrous
acid by the nitrite-forming bacteria is completed; this is due to the fact
that free ammonia, liberated from the interaction of the ammonium
salt and carbonate, has a toxic effect upon the nitrate organism. 6
The latter is present in crude culture together with the nitrite former
and, even when the latter reaches its maximum, the former is still
dormant. However, as soon as all the ammonia is transformed into
nitrite, the nitrate former becomes active. When transfers are made
from the crude culture, the stage of oxidation will account for the organ-
ism which will be prevalent. If the transfer is made at an early stage

18 Migula, W. Beitrage zur Kenntnis der Nitrifikation. Centrbl. Bakt. II,
6: 365-370. 1900.

19 Warington, 1878 (p. 63).

68 PRINCIPLES OF SOIL MICROBIOLOGY

of oxidation, when the ammonium salt is still abundant, the nitrate
former may be entirely eliminated in the process of several consecutive
transfers. If nitrite is substituted in the medium, in place of the
ammonium salt, and the culture is inoculated with soil or from a pre-
vious culture during the process of nitrate formation, and transfers are
constantly made upon the nitrite medium, the nitrite forming organism
may be entirely eliminated. The two bacteria can thus be separated
by means of their characteristic metabolism.

Solid media for the isolation and cultivation of the nitrite forming
organism. Silicic acid media were used first 20 for the isolation of the
nitrite forming bacterium.

Equal volumes of clear sodium or potassium silicate (specific gravity 1.05 to
1.06) andHCl (specific gravity 1.10) are mixed by pouring the first into the second;
the mixture is then dialyzed in parchment paper dialyzers, for several days in
distilled water, which is repeatedly changed. 21 When the dialyzate gives no
further reaction, other than mere turbidity, with AgN0 3 , the dialysis is com-
pleted. The clear solution contains about 2 per cent silicic acid and can be
preserved for three months in clean glass-stoppered bottles and readily sterilized
at 115° to 120°C. In addition to the silicic acid, four liquid solutions are pre-
pared :

1. (NH 4 ) 2 S04 3 grams 2. 2 per cent solution of ferrous

K 2 HPO 4 1 gram sulfate

MgS0 4 -7H 2 0.5 gram 3. Saturated NaCl solution

Distilled water 100 cc. 4. Milk of magnesia, i.e., a thick

suspension of finely powdered
magnesium carbonate in dis-
tilled water
Fifty cubic centimeters of the silicic acid solution is placed in a flask, then 2.5 cc.
of solution 1 are added and 1 cc. of solution 2. Enough milk of magnesia is added
to give the mixture a milky appearance (0.1 per cent sodium carbonate solution
may be used in place of the magnesia). The mixture is then poured, with con-
tinued stirring, into sterile, small thin-walled Petri dishes. Finally, one drop
of solution 3 is placed in the center of each plate. When allowed to rest in an
horizontal position, the liquid solidifies in about an hour. To get a more solid
medium, it is better to allow the dishes to rest 24 hours, then dry them out in the
thermostat.

The medium is inoculated either directly into the flask before the plates are
poured, or by placing a drop on the surface of the plate. The drop is spread over
the surface of the medium with the tip of a sterile glass rod and the same rod is
used for the inoculation of a second and third plate, so as to obtain a series of di-
lutions.

20 Winogradsky, 1891 (p. 65).

21 Omeliansky, 1899 (p. 66).

AUTOTROPHIC BACTERIA 69

The dishes are incubated in an inverted position at 25° to 30°C. The
development of an organism on the plate is first detected by chemical
tests for the formation of nitrous acid and disappearance of ammonia.
The tests are made by cutting a piece of the gel with a sterile loop
and placing them in dishes containing the proper reagent. The culture
can then be "fed" with 1 or 2 drops of a sterile 10 per cent ammonium
sulfate solution to bring about further development of the organism.

The minute microscopic colonies are better studied on the clear
(sodium carbonate) medium and soon after the addition of a fresh por-
tion of ammonium sulfate. The colonies are at first colorless, then they
become yellowish to brownish and finally dark brown; after a certain
time (10 to 14 days), the dark colonies clear up, beginning from the
edge toward the center, till finally the dark colony becomes colorless.
A pure colony will show uniform fine granulation up to the edge, while
contaminated colonies have an hyaline rim. A clear zone is formed
around each colony on the MgC0 3 medium due to the fact that the
latter is gradually dissolved by the nitrous acid; it is often difficult,
however, to demonstrate this zone. The plate is carefully examined
with a magnification of 50 to 100, and several clear surface colonies are
selected for transfer. Winogradsky recommended the use of finely
drawn sterile glass rods which are first dipped into the colony, then
transferred into the flasks with sterile liquid medium and, by striking
the bottom of the flask, the tip of the rod with the inoculum is broken
off and left in the flask. A number of transfers are made, since, in many
cases, the organism fails to develop. It is much more difficult to obtain
a culture from the dark colonies (zooglea) than from the colorless
colonies. To prove the purity of the culture, a few drops (about 0.5
cc.) of the liquid are inoculated into bouillon and meat peptone agar.
No growth should take place after two weeks incubation; this combined
with microscopic examinations indicates the absence of contaminations.

The silica gel can also be prepared by the method of Stevens and Temple, 22
as modified by Doryland: 23 24 grams of K 2 Si0 3 and 8.4 grams of Na 2 Si0 3
are dissolved in 500 cc. of water to give a concentration of 34.2732 grams of
H 2 Si0 3 per liter. One-half this concentration of H 2 Si0 3 per liter gives a
medium, which will solidify in approximately five minutes, thus making it
suitable for plating. The mixture of the two salts lessens the danger of too

22 Stevens, F. L., and Temple, J. C. A convenient mode of preparing silicate
jelly. Centrbl. Bakt. II, 21: 84 -87. 1908.

33 Doryland, C. J. T. Preliminary report on synthetic media. Jour. Bact. 1:
135-152. 1916.

70 PRINCIPLES OF SOIL MICROBIOLOGY

great a concentration of sodium. The detrimental influence of too great a con-
centration of the sodium and potassium salts can still further be lessened by
using a mixture of acids, such as equivalent solutions of HC1, H 2 S0 4 and H3PO4,
thus giving in the finished medium chlorides, sulfates and phosphates of both
bases.

The solutions of the three acids are standardized separately against the solu-
tion of the silicates, so that 1 cc. of each acid would just neutralize 1 cc. of the
silicate solution, against methyl orange. Methyl red and brom cresol purple
can also be employed. Before the final standardization, 0.5 gram MgSO.j, 0.01
gram CaC0 3 or CaO, 0.01 gram Fe 2 (SO 4)3 or FeCl 3 , 0.01 gramMnS0 4 and 1 gram
of ammonium sulfate are added to the HC1 solution.

The three acids are then mixed, taking 153.5 cc. of HC1, 77 cc. of H 2 S0 4 and
116 cc. of H3PO4. One cubic centimeter of this mixture will just neutralize 1 cc.
of the silicate solution, using phenolphthalein as an indicator. The two solutions
are placed in sterile bottles connected by siphons with automatic burettes, the
overflow caps of which are plugged with cotton to prevent contamination from
the air. The solutions are allowed to stand several hours, to sterilize the con-
tainers, then 5 cc. portions of the acid mixture followed by the same amounts
of silicate mixture are placed in sterile Petri dishes, which are then rotated
thoroughly; the jelly sets in five minutes. This medium has, however, never
been tested out sufficiently; it is possible that the osmotic concentration of the
salts may prove too excessive.

In addition to the silica plate method, three more methods are
available for the isolation of the nitrite forming bacteria.

Method 1. Agar may be prepared according to Beijerinck, 24 whereby 3 per cent
agar is dissolved in distilled water, filtered and allowed to solidify in thin layers
in glass containers. The solidified agar is then cut up into pieces, covered with
distilled water and incubated at 37° for two weeks; the soluble constituents of the
agar come into solution and are decomposed; the water is changed several times.
The agar is so purified that it can be used for the cultivation of the organisms.
It is preserved either under water or in a dried condition. Two-tenths per
cent NH4NaHP0 4 -4H 2 and 0.05 per cent KC1 and chalk are then added to the
agar and brought into solution. The plates have a milky appearance. The agar
may be washed in distilled water for several days, then dried at 60°C. A 2.5 per
cent solution of agar is then made, tubed in 10 cc. portions and sterilized in the
autoclave at 15 pounds pressure. Three solutions are then prepared and sterilized
in small portions.

grams in 100 cc.

1 . K 2 HPO 4 1.5

2. (NH 4 ) 2 S0 4 15

MgSO 4 0. 75

Fe 2 (SO<)3 0.02

u Beijerinck, W. M. Kulturversuche mit Amoben auf festem Substrate. Cen-
trbl. Bakt. I, 19: 257-267. 1896.

AUTOTROPHIC BACTERIA 71

3. NaCl 3.0

Na 2 C0 3 1.5

The agar is melted and cooled to 40°C. Portions (1 cc.) of the three solutions
are placed in sterile petri-dishes, inoculum is added, then the melted agar. All
the contents of the plate are properly mixed. If it is desired to use MgC0 3 ,
it should be added to the plate directly, at the time of pouring, and Na 2 C0 3
omitted from solution 3. The colonies of the bacterium develop on this medium
only slowly and a prolonged incubation period (3 to 4 weeks) is required. Agar
media are also more favorable to the development of contaminating organisms
than silica gel.

Method 2r. This is the magnesium carbonate — gypsum block method. 26,26 A
mixture is made of 300 grams gypsum (CaS0 4 -H 2 0), 30 grams MgC0 3 and 3 grams
MgNH 4 P04. This is carefully made into a homogeneous putty-like mass by
means of water or of a water extract of a fertile soil, using 250 grams per liter of
water, then mixing 8 parts of the powder and 3 parts of the liquid. The paste is
then put upon a glass plate and, by means of a knife, streaked out to a thickness
of 0.5 to 0.75 cm. Round portions are cut out for the plates and oblong for the
tubes. After the material has completely solidified, it is sterilized together with
the glass containers. A small amount of the sterile nutrient liquid, without the
ammonium sulfate and MgC0 3 , is placed at the bottom of the container, and the
surface of the plate is inoculated from the liquid culture. The nitrite forming bac-
teria develop on these blocks as yellow-brown colonies. MgC0 3 alone can be used,
to which the nutrient solution is added. The yellowish colonies sink into this
medium due to the dissolution of the MgC0 3 by the nitrous acid. The use of
filter paper, partly covered with the nutrient solution, in addition to some MgC0 3 ,
has also been suggested. 27 The organism forms minute yellow dots becoming
gradually brown. However, even the most recent students on this subject 28
found that the original silica gel method of Winogradsky is still the best for the
isolation of the nitrite forming bacteria.

Method 3. Winogradsky 29 recently suggested the possibility of using a
direct method for the isolation of nitrite forming, as well as other bacteria. This
new method promises to replace all the other methods, due to the rapidity with
which the organisms can be isolated. A series of plates containing silica gel,
prepared by one of the methods outlined above, and ammonium salt as the sole
source of energy are inoculated by placing minute particles of soil into the gel all
over the plates; these are then incubated. The nitrite-forming bacteria develop

25 Omeliansky, W. L. Magnesia-Gipsplatten als neues festes Substrat fur die
Kultur des Nitrifikationsorganismen. Centrbl. Bakt. II, 6: 652-655. 1899.

26 Makrinov, J. Magnesia-Gipsplatten und Magnesia-Platten mit organischer
Substanz als sehr geeignetes festes Substrat fur die Kultur der Nitrifikations-
organismen. Centrbl. Bakt., II, 24: 415-423. 1910.

27 Omeliansky, W. L. Kleinere Mitteilungen uber Nitrifikationsmikroben.
Centrbl. Bakt., II, 8: 785-787. 1902.

28 Bonazzi, A. On nitrification. III. The isolation and description of the
nitrate ferment. Bot. Gaz. 68: 194-207. 1919.

29 Winogradsky, 1925 (p. 7, 11).

72 PRINCIPLES OF SOIL MICROBIOLOGY

in the form of a zooglea-like zone around the soil particles, and can be readily
isolated by transferring to sterile liquid medium.

Morphology of the nitrite forming bacterium. In the process of nitrifica-
tion we are dealing not with one organism, but with a group of closely
related organisms. One strain was isolated 30 from soils of Western
Europe and was called Nitrosomonas (Nitr. europca Winogradsky) .
Another strain was isolated from soils of South America (Campinas-
Brazil, Quito-Ecuador) and Australia (Melbourne) and was called
Nitrosococcus.

When a vigorous culture of Nitrosomonas is inoculated into the sterile
liquid medium, an appreciable nitrite reaction is obtained in 2 to 3
days, reaching a maximum in 5 to 6 days. When the culture is
examined microscopically, very few organisms are found in the super-
natant liquid, but rare, compact, variable (10 to 50/x) zooglea recognized
with difficulty are formed in the sediment. When a drop of KI-I
solution is added, the cells are easily recognized. In 8 to 10 days, the
liquid becomes opalescent and, on examination in a hanging drop, it
is found to consist of swarming, ellipsoidal, motile microbes, as seen in
Plate III. This shows the zooglea broken up into a swarm stage.

The cells of the Nitr. europea are always oblong, similar to a zero,
never coccus-like, 1.2 to 1.8/j. long by 0.9 to 1/j, wide. They can be
stained with all ordinary basic anilin dyes and are Gram positive.
The motile cells of the swarm carry on one end a moderately long

PLATE III
Nitrifying Bacteria

5. Surface colonies of Nitrosomonas on silicic acid gel; stained with carbol
fuchsin, X 130 (from Gibbs).

6. Surface colony of Nitrosomonas on silicic acid gel; stained with carbol
fuchsin, X 800 (from Gibbs).

7. Nitrosomonas europea, X 660 (from Winogradsky).

8. Nitrosomonas javanensis, X 660 (from Winogradsky).

9. Colonies of Nitrobacter; deep-seated colonies on washed agar, unstained,
X240 (from Gibbs).

10. Nitrobacter from culture in liquid medium; stained with carbol fuchsin,
X 1660 (from Gibbs).

11. Nitrobacter from nitrite-agar cultures, 2 months old (from Fred and Daven-
port).

12. Nitrobacter from nitrite-agar cultures, 15 days old, showing polar flagella
(from Fred and Davenport).

30 Winogradsky, 1892 (p. 62).

PLATE III

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AUTOTROPHIC BACTERIA 73

flagellum; the latter can be stained by the Loeffler method, by adding
10 to 15 drops of 1 per cent sodium carbonate solution to the ferro-
tannate, or by the method of Zettnow.

In some cases the motile stage may be predominant over the zooglea
formation or vice versa. It is important to note that the predominance
of the particular stage is characteristic of the strain, so that one might
suspect that we are dealing here with two distinct races. The two
stages are also distinguished by their rapidity of oxidation of ammonia,
the motile stage being the stronger. Winogradsky suggested that the
cause for this lies in the fact that the active stage (monas) consumes
more energy and comes more readily in contact with the ammonia and
oxygen than the non-motile zooglea. The zooglea are probably resting
stages, being also more resistant to drying. Gibbs isolated the Nitro-
somonas from soils of North America and found it to be 1.2 to 1.5 by
0.9 to l.Ofj. in size, rounded or oval, which stained uniformly. The
organism was found chiefly in the free cell stage. Thermal death point
of the organism was between 53° and 55°C. (10 minutes).

Other organisms also capable of oxidizing ammonia to nitrite, but
having different morphological characters, were isolated by Winogradsky
from soils in Europe. So, for example, the form isolated from soil of
St. Petersburg was a true coccus, about 1/jl in diameter, sometimes form-
ing zooglea and sometimes growing free, but never in the swarm stage.
A constant property of its morphology is a central nucleus-like body,
made visible by various stains, particularly by methylene blue.

The Nitr. javanesis is a still smaller coccus (0.5 to 0.6/x) and has been
isolated by Winogradsky from the soil of Buitenzorg, Java. This form
grows both in the monas and zooglea stages, the first having very long
(up to 30m) flagella. Free cells are present only in the swarm stage,
mostly in pairs. Winogradsky also isolated from the soil of Quito,
South America, a non-motile coccus (Nitrosococcus) termed by Migula
Micr. nitrosus. This organism consists of large cocci 1.4 to 1.7/x in
diameter, always growing as free cells and never forming zooglea,
while the motile stage could never be demonstrated. It is obli-
gate aerobic, gram-positive. It forms rather large, opaque yellowish
colonies on the silica gel but the colonies are made up of free cells.
The cells appear larger in the living state than in the stained prepara-
tion, probably due to a thick gelatinous membrane which does not
stain or becomes invisible on desiccation. Bonazzi 31 isolated the

"Bonazzi, 1919 (p. 71).

74 PRINCIPLES OF SOIL MICROBIOLOGY

Nitrosococcus also from the soil of North America (Ohio) : it forms small
yellowish colonies (224 by 160,u on the silica gel) surrounded by a
colorless halo, due to the solution of the MgC0 3 . Colonies of 1 mm. in
diameter have been obtained by renewing the (NH 4 ) 2 S0 4 in the plates
when necessary. The organism was found to be present in two stages:
(1) as megalococci, 1.25/* in diameter, of a slightly irregular form, oc-
curring in thick gelatinous masses; when the cultures are in the process
of active oxidation, the megalococci give rise to small cocci; (2) a form
which leaves the gelatinous mass and become free. The hanging drop
cultures showed no motility.

The organism is stained as follows. The cover glass preparation is
fixed in flame, treated with mordant 1 minute in the cold with a 0.25
per cent solution of malachite green in distilled water, washed with
cold water, and stained cold with a 0.25 per cent water solution of
gentian violet for another minute. The stain is then rapidly washed
with water previously heated to 50 to 60°C. The megalococci are
stained deep purple and the small cocci purple-black.

The existence of the several forms of nitrite-forming organisms in
the soils from different continents was explained by Winogradsky as due
to the probability that local conditions favored the adaptation of a
particular variety.

Nitrate forming bacteria (Nitrobacter) . The organism that is able
to oxidize nitrite to nitrate was discovered by Winogradsky 32 in 1891
in the solutions where nitrate formation was taking place.

It was finally cultivated in a sodium nitrite medium to which the ordinary
nutrients and magnesium carbonate or sodium carbonate have been added as
follows :

NaN0 2 1.0 gram NaCl 0.5 gram

K2HPO4 0.5 gram Na 2 C0 3 (anhydride) . . 1.0 gram

MgS0 4 0.3gram FeS0 4 0.4gram

Distilled water 1000 cc.

(The ferrous sulfate may be reduced to a trace and the anhydrous Na2C0 3 to
0.5 gram.)

Fifty cubic centimeter portions of the sterile solution in flasks are inoculated
with soil. The course of the reaction is followed by the disappearance of the
nitrous acid and the appearance of nitric acid (using diphenylamine and concen-
trated sulfuric acid). When all the nitrite has been oxidized, fresh portions of
the salt are added, in the form of a sterile solution as in the case of the ammonium
sulfate, and the culture is studied microscopically, using carbol fuchsin for
staining.

32 Winogradsky, 1904 (p. 64).

AUTOTROPHIC BACTERIA 75

The inoculated solutions show no turbidity or pellicle formation. It
is only after repeated additions of nitrite that a bluish slime can be
distinguished on the bottom and wall of the flask wherever it is in con-
tact with liquid. When this slime is examined microscopically, it is
found to consist of a layer of minute, spindle-shaped (generally) rods
staining with difficulty. After several transfers into fresh flasks con-
taining sterile liquid medium, the culture is sufficiently enriched, so that
plates can be prepared.

The following agar medium is used for the isolation and cultivation of the
organism:

NaN0 2 2.0 grams Agar 15.0 grams

Na 2 C0 3 (anhydride) ... 1.0 gram Tap water 1000 cc .

K 2 HP0 4 0.5 gram

The surface of the solidified agar plate is smeared with a drop of the solution
in which the organism has developed, and the plates are allowed to incubate 14
days at 30°. The streak then appears opaque and rounded numerous small drop-
lets are differentiated with the naked eye. The sub-surface colonies are shining,
slightly brownish, of various shapes developing in two weeks to a diameter of
30 to 50/x. On the surface of the plate, the colonies appear as round homogeneous
drops, reaching, in two weeks, a diameter of 100 to 180/*. On slants the growth is
dirty white, with a large, semi-fluid drop at the bottom. When a loop of this ma-
terial is transferred into a 25 cc. portion of nitrite solution, the nitrite reaction
disappears in 3 to 4 days.

Care should be taken not to mistake other bacteria for the nitrate
former, since several soil bacteria grow on this medium. The smallest
colonies are selected and carefully checked up with the disappearance
of the nitrite reaction, combined with prolonged incubation (3 weeks at
30°C). Transfer is then made of characteristic colonies by means of the
open capillary glass rods described above. From a series of transfers,
some will be found to develop into pure cultures. To ascertain the
purity of the culture, several drops of the liquid culture are added
to bouillon or agar.

Nitrobacter is a non-motile, rod-shaped bacterium, obligate aerobic,
non-spore forming, gram-negative. The cells are stained with carbol
fuchsin, and can then be washed with dilute acidified alcohol. It is 1 by
0.3 to 0.4/z in size, with one or both ends pointed; the staining is not uni-
form, the central part being stained, while the pointed ends remain almost
colorless. In Loeffler's alkaline methylene blue, only the nucleus-like
bodies are stained well, but not the surrounding cell. By staining in
warm gentian-violet, then washing with a 2 per cent NaCl solution, the
cells are found to be 1.2 to 1.5 by 0.6 to 0.7^, surrounded by a capsule,

76 PRINCIPLES OF SOIL MICROBIOLOGY

commonly found in single cells or in pairs. The thermal death-point is
56° to 58°C. The colonies on agar are rounded and light-brown, in 7 to
10 days at 28°C. After two weeks, they are darker in color; deep
colonies are 30 to 50 /j. in diameter and surface colonies are 50 to 150ju
in diameter, with a tendency to spread. The nitrate bacteria may also
be isolated directly from the silica gel plate, when nitrite is used as the
only source of energy.

Beijerinck 33 claimed that the nitrate-forming bacterium can grow in
the presence of various organic substances but that the organism loses
some of its power of oxidation after growing in the presence of soluble
organic matter. Accordingly, he suggested that growth and oxidation
are two distinct functions and that even if small amounts of organic
substances (0.05 per cent) did not prevent the oxidation of nitrite to
nitrate, reproduction of the organism in solutions containing large
amounts of these substances caused a stable modification in their
physiology.

However, Fred and Davenport 34 obtained no evidence to support the
statements of Beijerinck. Contrary to the general opinion, they found
that certain forms of organic matter benefit rather than injure the
nitrate-forming bacterium. They grew the Nitrobacter on washed
nitrite agar and on slants of Niihstoff-Heyden agar with or without
N0 2 -ion present. The organism does not reproduce in Nahrstoff-
Heyden solution, which is non-toxic, while beef-infusion and peptone-
beef infusion, in higher concentrations, are toxic. The harmful ma-
terial is non-volatile and can be removed by extraction with ether
or alcohol. Nitrobacter will live 2 to 6 weeks in 1 per cent solutions of
Nahrstoff-Heyden, gelatin, peptone, casein, yeast water and milk,
and also in distilled water without any further development. Gelatin,
peptone, casein, skimmed milk, beef infusion and beef extract do not
decrease the oxidation of nitrite by the organism; asparagine,
(NH 4 )oS0 4 , and urea retard the oxidation; Nahrstoff-Heyden increases
it above water controls; sealed agar slants of Nitrobacter were kept
more than a year without serious injury to their power of oxidation.

Winogradsky 35 definitely pointed out that growth and nitrite oxida-

33 Beijerinck, M. W. tlber das Nitratferment und iiber physiologische Art-
bildung. Folia Microb. 3: 91-113. 1914.

34 Fred, E. B., and Davenport, A. The effect of organic nitrogenous com-
pounds on the nitrate-forming organism. Soil Sci. 11: 389-407. 1921.

36 Winogradsky, S. Sur la pretendue transformation du ferment nitrique en
espece saprophyte. Compt. Rend. Acad. Sci. 175: 301-303. 1922.

AUTOTROPHIC BACTERIA 77

tion are inseparable functions; organic matter (1 per cent peptone) may
paralyze the organism, but does not kill it and does not change it, since,
when transferred upon proper media, it resumes its activities.

The respiration of the organisms, the chemistry of the processes of
nitrite and nitrate formation, and their importance in soil fertility
are discussed in detail elsewhere (p. 525).

Occurrence of nitrifying bacteria in the soil. All soils, not very acid in
reaction, contain bacteria capable of oxidizing ammonium salts to
nitrites and the latter to nitrates. The limiting acidity for the develop-
ment of these bacteria in the soil is pH 4.0 to 4.4, while the optimum
reaction is at pH 6.8 to 7.3. 36 When a soil more acid in reaction than
the minimum for their development is treated with lime, the or-
ganisms will gradually appear in the soil; however, inoculation with
a good fertile soil is often practiced, so as to introduce the or-
ganisms immediately. The numbers of the nitrifying bacteria per
gram of soil vary from a few to 10,000. 37 The method commonly used
for this determination consists in diluting the soil with sterile water,
then adding 1 cc. portions of the various dilutions to the proper media.
Positive growth indicates a minimum number of organisms. It is
possible, however, that many cells have to be added to a liquid medium,
before growth can take place, since conditions are not made as favorable
for their development in artificial liquid media as in normal soil. In
humid soils, the bacteria are present in the upper few inches and
rapidly disappear in the subsoil. However, in arid soils, they occur to
a depth of many feet. 38

In addition to the typical nitrite and nitrate bacteria described by
Winogradsky and isolated by other investigators, various reports have
been made concerning the isolation of nitrite and nitrate forming
organisms, ranging from typical autotrophic forms, like the nitro-
microbium of Stutzer and Hartleb, 39 to forms possessing properties
altogether uncharacteristic of autotrophic bacteria, such as cellulose
decomposition, gelatin liquefaction, nitrate reduction, etc.; 40 however,
the last investigations still remain to be verified.

36 Gaarder, T., and Hagem, O. Nitrifikation in sauren Losungen. Bergeng
Museum Aarbook. 1922-3, No. 1.

"Duggeli, 1923 (p. 39).

38 Lipman, 1912 (p. 36).

39 Stutzer, A., and Hartleb, R. Untersuchungen fiber die bei der Bildung von
Salpeter beobachteten Mikroorganismen. Mitt, landw. Inst. Breslau, 1: 75-99,
197-232. 1901.

"Sack, J. Nitratbildende Baktcrien. Centrbl. Bakt., II, 62: 15-24. 1924;
64: 32-37, 37-39. 1925.

78 PRINCIPLES OF SOIL MICROBIOLOGY

Bacteria deriving their energy from the oxidation of sulfur and its com-
pounds. The sulfur bacteria do not form any unifonn group of micro-
organisms , as in the case of the nitrifying bacteria, either morphologi-
cally or physiologically. Morphologically they are found among the
Desmobacteriaceae (Thiobacteriales) and among the Bacteriaceae
(Eubacteriales). Physiologically they may oxidize hydrogen sulfide
and other sulfides, elementary sulfur, or thiosulfate and they may act
either in an acid or in an alkaline reaction. Some are obligate auto-
trophic and some are facultative. The bacteria which are found in
normal, fertile soils or those that become active in the soil, when intro-
duced, are limited chiefly to the genus Thiobacillus among the Bac-
teriaceae.

All microorganisms require minute quantities of sulfur for the syn-
thesis of their protoplasm. Various bacteria and even some fungi seem
to be capable of oxidizing small amounts of sulfur. But only certain
bacteria work over much larger quantities of sulfur than would be neces-
sary for their body structure, since they utilize the sulfur or its com-
pounds as a source of energy. The sulfur is to the sulfur bacteria, as

PLATE IV
Sulfur and Iron Bacteria

13. Beggiatoa alba, thread forming sulfur-oxidizing bacterium: a, in liquid
culture rich in H 2 S; b, culture kept 24 hours in liquid freed from H 2 S; c, 48 hours
later in the same liquid (sulfur droplets have disappeared, cell division takes
place and protoplasmic contents are left), X 600 (after Omeliansky).

14. Thread-forming, sulfur-oxidizing bacteria: (x) Beggiatoa media and (y)
Beggiatoa minima, X 600 (after Omeliansky).

15. Thioploca ingrica, X 200 (after Wislouch and Omeliansky).

16. Young threads of Thiothrix nivea, X 600 (after Omeliansky).

17. Thiophysa macrophysa, showing drops of sulfur on periphery and oxalate
crystals in center, X 660 (after Nadson and Omeliansky).

18. Thiospirillum winogradskii: a X 100 and b X 660 (from Omeliansky).

19. Chromatium okcnii, X 660 (after Omeliansky).

20. Achromatium oxaliferum: A, showing the calcium bodies, but not sulfur;
B, without the calcium bodies, but with a number of droplets of sulfur (from
Nadson and Wislouch).

21. Thiobacillus thioparus, showing drops of precipitated sulfur among the rod-
shaped organisms, X 1000 (from Diiggeli).

22. Thiobacillus thiooxidans, X 660 (Original). ■

23. Diagrammatic sketch of several typical iron bacteria: a, Spirophyllum fer-
rugineum; b, Gallionella ferruginea; c, Leplothrix ochracea, X about 720 (from
Harder, by courtesy of U. S. Geological Survey).

24. Cladolhrix dicholoma, X 190 (after Molish).

PLATE IV

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21

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22

23

24

AUTOTROPHIC BACTERIA 79

the ammonium sulfate is to the Nitrosomonas and Nitrosococcus, the
nitrous acid and nitrite to Nitrobacter and the carbon compounds to the
heterotrophic bacteria.

The sulfur bacteria, or those bacteria which are capable of obtaining
the energy necessary for their growth from the oxidation of sulfur or its
compounds, should be distinguished from other bacteria taking part in
the sulfur cycle, such as those liberating H 2 S in the hydrolysis of pro-
teins or in the reduction of sulfates.

Classification of sulfur bacteria. The sulfur oxidizing bacteria can
be divided into five groups.

1 . Thread-forming, colorless bacteria, accumulating sulfur within their
cells; Beggiatoa and Thiothrix are representatives of this group.

2. Non-thread forming, colorless bacteria, accumulating sulfur within
their cells; a number of forms (Thiospirillum, Thiovulum, Achromatium,
etc.) of various sizes and shapes, the distinguishing characteristic of
which is the fact that they oxidize H 2 S and accumulate sulfur within
their cells, are included in this group. Some of these have been isolated
in pure culture.

3. Purple bacteria. Some of these seem to play a part in the sulfur
cycle, although none of the sulfur forms have yet been isolated in pure
culture.

4. Colorless, non- thread forming sulfur oxidizing bacteria, which do
not accumulate sulfur within their cells, but which produce an abun-
dance of sulfur (from H 2 S and thiosulfates) outside of their cells. The
two characteristic and most important forms belonging to this group are
Thiobacillus denitrificans Beijerinck, an anaerobic form, deriving its
oxygen from the decomposition of nitrates, and Thiobacillus thioparus
Beijerinck, which oxidizes thiosulfates, H?S, and S and allows an exten-
sive accumulation of sulfur from the first two.

5. The fifth group is similar to group 4 in morphology (but is less than
1/jl in length) and is distinctly different physiologically. The organisms
belonging to this group can act upon thiosulfates and H 2 S, but
they oxidize elementary sulfur very rapidly, allowing the medium to
become acid up to a reaction of pH 0.6 to 1.2. The only known
representative of this group is Thiobacillus thiooxidans Waksman and
Joffe.

The sulfur bacteria can also be divided into, a, sulfide bacteria, or those
organisms which act primarily upon H 2 S and sulfides and which would
include the first three groups in the previous classification; b, thiosulfate
or "thionic acid" bacteria, equivalent to group 4; c, sulfur bacteria,

80 PRINCIPLES OF SOIL MICROBIOLOGY

acting primarily upon elementary sulfur, equivalent to group 5. The
first four groups have their optimum on the alkaline side of neutrality,
while the last group has its optimum on the acid side of neutrality.
Only the last two groups (4 and 5) occur in the soil and play an active
part in the oxidation of sulfur and its compounds in the soil. The
first three groups do not occur to any extent in normal cultivated soils,
but are mentioned here for the sake of completeness.

Group I. Colorless, thread-forming sulfur hacteria, accumulating sulfur
within their cells (Nos. 13-16, PI. IV). The representatives of this
group are able to act only upon H 2 S. In view of the fact that these
are primarily water and mud forms, only the general principles in-
volved are discussed. This group of sulfur oxidizing bacteria consists
of three genera : Beggiatoa, including motile organisms forming no sheaths ;
Thiothrix, fastened forms forming no sheaths; and Thioploca, thread-
forming bacteria, surrounded with a jelly-like sheath. The Beggiatoa
were the first organisms to attract attention as having to do with the
oxidation of sulfur or its derivatives. Cramer 41 pointed out that the
granules found within the cells of Beggiatoa consisted of sulfur. Cohn 42
then proposed the theory that the Beggiatoa and the purple bacteria
produce hydrogen sulfide by the reduction of sulfates. But it was
Winogradsky 43 who demonstrated that the hydrogen sulfide is pro-
duced by other bacteria and is oxidized by the Beggiatoa to sulfur and
sulfuric acid.

This oxidation is so important for the very existence of these organ-
isms that, when the hydrogen sulfide is taken out of the medium, they
oxidize the sulfur present within their cells and, when this is used up,
they die out. The energy liberated in this process is utilized by the
organisms for the assimilation of carbon dioxide. For every gram of
carbon, 8 to 19 grams of sulfur are consumed. If there is enough H 2 S,
the presence of traces of organic substances and nitrates in the water is
sufficient for the development of these organisms, while the presence of
sugars, peptone and like nutrients will stimulate the growth of other
microbes but will injure these sulfur bacteria.

41 Cramer, In Midler, C, Chemisch-physikalische Beschreibung der Thermen
von Boden in der Schweiz. 1870.

42 Cohn, F. Untersuchungen liber Bakterien II. Beitr. Biol. Pflanz. 1: H. 3,
p. 141. 1875.

43 Winogradsky, S. Beitrage zur Morphologie und Physiologie der Bakterien.
I. Schwefelbakterien. Leipzig. 1883; Ann. Inst. Past. 3: 1883, No. 2; Uber
Schwefelbakterien. Bot. Ztg. 45: 489, 513, 529, 545, 569, 585, 606. 1887.

AUTOTROPHIC BACTERIA 81

According to Winogradsky, the sulfuric acid formed is neutralized
by the calcium carbonate or bicarbonate present in the water, since the
reaction of the water cultures of these bacteria was not found to become
acid. In reference to the physiology of these organisms, the results of
Winogradsky can be summarized as follows: (1) The sulfur bacteria
oxidize hydrogen sulfide and accumulate sulfur in the form of small
spheres, consisting of soft amorphous sulfur which never crystallizes
in the living cells. (2) They oxidize the sulfur to sulfuric acid, which
is at once neutralized, by the carbonates present, into sulfates. (3)
Without sulfur, the organisms soon die off. (4) They can live and
multiply in liquid containing only traces of organic substances.

This last point was refuted by Keil, 44 who demonstrated that the
organisms are autotrophic and do not need organic substances for their
growth. Keil claims to have isolated pure cultures of Beggiatoa and
Thiothrix, and found that these organisms are capable of living in
media free from any traces of organic matter, although the presence of
small quantities of organic substances is not detrimental to them.
The raw cultures were obtained by Keil by placing a layer of black mud
containing these bacteria on the bottom of a glass container, 3 to 4 cm.
high, covering it with 2 to 3 cm. of river water and placing in the dark,
at room temperature. The Beggiatoa formed a white layer over the
mud. The Thiothrix could be easily distinguished by the fact that they
were fastened at one end. By adding water from a sulfur spring to
Petri dishes, then placing these under a bell-jar, the amount of gas
necessary for the growth could readily be ascertained. Ammonium
salts were found to be used as sources of nitrogen and only carbonic
acid as a source of carbon. Carbon dioxide pressure may vary within
the limits of 0.5 and 350 mm. (25 mm. is the optimum) ; oxygen may
vary within 10 to 20 mm., and H 2 S within 0.6 to 1.7 mm. The presence
of carbonates is important for the neutralization of the acids.

The pure cultures were obtained from the enriched culture by the
mere mechanical process of washing out all other organisms first with
ordinary water and then with sterile water. This was followed by
growth under the bell-jar, at definite gas pressures and frequent changes
of medium. Further information on this group of organisms is found

44 Keil, F. Beitriige zur Physiologie der farblosen Schwefelbakterien. Beitr.
Biol. Pflanz, II: 335-372. 1912.

82 PRINCIPLES OF SOIL MICROBIOLOGY

in the work of Omeliansky, 45 Duggeli, 46 Bavendamm, 47 and others. 48
The Thioploca has been studied in detail by Wislouch 49 and Kolkwitz. 50
Group II. The second group of the sulfur oxidizing bacteria is
heterogeneous in nature and consists of all colorless organisms, ivhich
form no threads and which contain sulfur within their cells. These also
act only on H 2 S (Nos. 17-20, PI. IV). This group includes organisms
of various forms. They may be obtained by placing the cut rhizomes
of water plants, together with the mud, into tall glass cylinders with
some river or canal water and a few grams of calcium sulfate. When
placed in the dark, H 2 S will be produced in 5 to 10 days, and the color-
less non-thread-forming sulfur bacteria are found in 3 to 6 weeks. The
nature of the plant or animal material, nature of the mud and quantity
of H 2 S produced, determine which species will predominate. Among the
organisms described at various times we might mention : Monas mulleri
and Monas fallax Hinze, 48 Thiophysa volutans Hinze, 51 Thiospirillum wi~
nogradskii Omeliansky, 52 Thiovulum Hinze, Spirillum Molisch, Bacterium
bovista (2 to 4 by 0.6 by l.Oju), Bacillus thiogenes (2 to 6 by 0.9 to 1.34;u),
and Achromatium. 53M Most organisms belonging to this group have
been found in water and in mud; few of them have been obtained in
pure cultures. They play an important part in the formation of the

46 Omeliansky, W. L. Der Kreislauf des Schwefels. Lafar's Handb. techn.
Mykol. 3: 214-244. 1904.

46 Duggeli, M. Die Schwefelbakterien. Neujahrsbl. Naturf. Gesell. Zurich.
1919, No. 121, 43 p.

47 Bavendamm, W. Die farblosen und roten Schwefelbakterien des Siisz- und
Salzwassers. G. Fischer. Jena. 1924.

4S Hinze, G. Beitrage zur Kenntnis der farblosen Schwefelbakterien. Ber.
deut. bot. Gesell. 31: 189-202. 1913. Molisch, H. Neue farblose Schwefel-
bakterien. Centrbl. Bakt. II, 33: 55-62. 1912.

49 Wislouch, S. M. Thioploca ingrica nov. spez. Ber. deut. bot. Gesell. 30:
470-473. 1912.

60 Kolkwitz, R. t)ber die Schwefelbakterie Thioploca ingrica Wislouch.
Per. deut. bot. Gesell. 30: 662-666. 1912.

61 Hinze, G. Thiophysa volutans, ein neues Schwefelbakterium. Ber. deut.
bot. Gesell. 21: 309-316. 1903.

62 Omeliansky, W. L. Tiber eine neue Art farbloser Thiospirillen. Centrbl.
Bakt. II, 14: 769-772. 1905.

63 Nadson, G. A. On the sulfur bacteria of the sea of Hapsala. Bull. Jard.
Bot. St. Petersburg, 13: 106-112. 1913; On the sulfur bacteria Thiophysa and
Thiosphaerella. Jour. Microb. (Russian) 1: 52-72. 1914.

64 West, G. S., and Griffith, B. M. The lime-sulfur bacteria of the genus Hil-
lowia. Ann. Bot. 27: 83-91. 1913.

AUTOTROPHIC BACTERIA 83

curative muds. 55 According to Nadson, some of these organisms, like
Achromatium and Thiophysa, can accumulate in their cells sulfur as
well as oxalate crystals. Jegunow described two sulfur bacteria:
Thiobacterium a, a motile, colorless, slightly curved organism, 4.5 to 9ju
long and 1.4 to 2.3ju wide, containing a finely granulated plasma and
large sulfur granules, and Thiobacterium /3, motile, colorless, curved,
2.5 to 5 by 0.6 to 0.8/x, and containing a row of shining sulfur granules.
Various bacteria belonging to this group have been reported 56 to occur
in the soil, namely: Spirillum agilissimum filled with black sulfur granules,
measuring about 6 to 10 by 1.8 to 2.0^, having rapid motility, and
isolated from river mud in Gratz; Chromatium cuculliferum which is
round to slightly elliptical, 6 by 4/j, of a slow motility, with black,
shining, sulfur drops always found in one pole, with one flagellum on the
granule-free pole. This latter form was found in rotting mass of algae
in the garden basin at Gratz. However, since none of the forms has
been considered from the point of view of its role in soil transformations,
their importance in the soil is doubtful. A detailed study of the
morphology and biology of Achromatium oxalifcrum Schew., containing
granules of a calcium salt (oxalate, carbonate or thiosulfate) and sulfur
has been made by Nadson and Wislouch. 57

Group III. This group consists of the sulfur oxidizing organisms
found among the purple bacteria. They are distinguished from the
sulfur bacteria described above by the production of a red, red violet
or red brown pigment which is unevenly distributed throughout the
cell; in addition to the red pigment (bacterio-purpurin), there is also
present in all these bacteria a green pigment (bacterio-chlorin). These
bacteria are found abundantly in sulfur springs and in mud waters.
Not all the purple bacteria are able to utilize hydrogen sulfide and not
all of them accumulate sulfur within their cells. Molisch 58 succeeded
in cultivating some of them in pure culture, but not the sulfur forms.
The role of sulfur in the metabolism of the purple bacteria is still an
open question, since, according to Molisch, the hydrogen sulfide is not

66 Jegunow, M. Bakterien Gesellschaften. Centrbl. Bakt. II, 2: 11-21, 441-
449; 478^82; 739-752. 1896.

56 Gicklehorn, J. Uber neue farblose Schwefelbakterien. Centrbl. Bakt. II,
50: 415-427. 1920.

87 Nadson, G. A., and Wislouch, C. M. La structure et la vie de la bacterie
geante Achromatium oxalijerum Schew. Bull. Jard. Bot. Rep. Russe. 22: 1-24.
1923.

68 Molisch, H. The Purpurbakterien nach neuen Untersuchungen. Jena.
G. Fischer. 1907.

84 PRINCIPLES OF SOIL MICROBIOLOGY

required for nutrition. These results are in direct opposition to the
earlier ideas of Winogradsky and others.

Group IV. This group includes the colorless organisms that do not
accumulate sulfur within their cells, but produce sulfur abundantly from
thiosulfate and hydrogen sulfide outside of their cells (No. 21, PI. IV).
These were first demonstrated by Nathanson 59 (1902) in sea water.
They were found to be able, by means of oxidation of hydrogen sulfide
or sodium thiosulfate, to reduce carbonic acid and construct organic sub-
stances from it. Nathanson used a medium of the following composi-
tion:

Na 2 S 2 3 2-10 grams NaCl 30.0 grams

KN0 3 1 gram MgC0 3 some

Na 2 HP0 4 0.5 gram Water 1000 cc.

MgCl 2 2.5 grams

A good growth of these bacteria was obtained after 1 to 2 days, in the
form of a white pellicle covering the surface; this consisted of rod-
shaped organisms intermixed with amorphous sulfur.

On adding agar to the above medium Nathanson has been able to
isolate the organism in pure culture. In the absence of the carbonate,
but in the presence of air containing carbon dioxide, the growth was
much slower. In the absence of both carbonate and carbon dioxide,
no growth took place, even in the presence of various organic substances.
The medium did not become acid even in the absence of carbonate.
While no sulfur accumulated within the cell, there was an abundant
production of free sulfur outside of the cell, not in direct contact with
the colony but at some distance from it. This led to the theory of
extracellular oxidation. Nathanson suggested that the sulfur is pro-
duced in a secondary reaction between the undecomposed thiosulfate
and the tetrathionate formed from the oxidation of the thiosulfate.

Beijerinck 60 employed the following medium:

Na 2 S 2 3 -5H 2 5.0 grams NH 4 C1 0.1 gram

NaHC0 3 l.Ogram MgCl 2 O.lgram

Na 2 HPO < 0.2 gram Water 1000 cc.

The medium was left unsterilized and was inoculated with canal water
and incubated at 28°to 30°C. In 2 to 3 days, the surface of the medium

69 Nathanson, A. tJber eine neue Gruppe von farblosen Schwefelbakterien
und ihren Stoffwechsel. Mitt. Zool. Station, Neapel 15: 655. 1902.

60 Beijerinck, M. W. Uber die Bakterien, welche sich im Dunkeln mit Kohlen-
saure als Kohlenstoffquelle ernahren konnen. Centrbl. Bakt. II, 11: 593-599.
1904.

AUTOTROPHIC BACTERIA 85

became covered with free sulfur, intermixed with bacteria. On making a
transfer into a fresh flask with medium, a sulfur layer was obtained in
24 hours, originating from the thiosulfate. This reaction is exothermic
and functions as a source of energy. The energy is used for the reduc-
tion of NaHC0 3 and for the building of the bacterial body. Calcium
sulfide, hydrogen sulfide and tetrathionate can replace the thiosulfate.
The ammonium salt can be replaced by nitrates. None of the
organic substances tested could replace the carbonic acid as a source of
carbon. The organism, Thiobacillus thioparus Beijerinck, was reported
to be a short rod, 3 by 0.5ju, not forming any spores, very motile and
very sensitive, so that on plates the organisms die off in a week.

By adding 2 per cent agar to the above medium the organism can be
grown on the plate; transfers are then made from individual colonies
into fresh lots of the liquid medium giving a pure culture of the organism.
The colonies are of a pin-point form and are distinguished from con-
taminations by their yellow appearance, due to an abundant separation
of sulfur. According to Duggeli, 61 this organism is only 0.3 to 0.5/x long.
Jacobsen 62 demonstrated that this organism can also oxidize sulfur to
sulfuric acid, in the following medium:

K0HPO4 0.5 gram CaCOj or MgCO s . . . . 20.0 grams

NELC1 ' 0.5 gram Precipitated sulfur. . . 10.0 grams

MgCl 2 0.2 gram Distilled water 1000 cc.

An organism similar to the Thiobacillus thioparus was found 63 to be
active in the oxidation of sulfur in alkali soil, giving the gross micro-
scopic reactions of the form studied by Nathanson and Beijerinck.

Group IV of the sulfur bacteria includes, in addition to the aerobes,
anaerobic bacteria which are able to obtain their oxygen from nitrates.
Beijerinck obtained an oxidation of sulfur accompanied by a reduction
of the nitrate to atmospheric nitrogen by using the following medium
in closed flasks and incubating at 30°C.

KNO3 . 0.5gram CaC0 3 20.0 grams

Na 2 C0 3 0.2 gram Sulfur 100.0 grams

K 2 HP0 4 0.2 gram Canal water 1000.0 cc.

61 Duggeli, 1919 (p. 82).

62 Jacobsen, H. C. Die Oxidation von elementarem Schwefel durch Bakterien.
Folia Microb. 1: 487-496. 1912; 3: 155-162. 1914.

63 Waksman, S. A. Microorganisms concerned in the oxidation of sulfur in the
soil. V. Bacteria oxidizing sulfur under acid and alkaline conditions. Jour.
Bact. 7: 609-616. 1922.

86 PRINCIPLES OF SOIL MICROBIOLOGY

The sulfur is oxidized to sulfuric acid which acts upon the CaC0 3
giving CaS0 4 and C0 2 . Beijerinck isolated, in pure culture, an organ-
ism, Thiobacillus denitrificans, which is a very motile, short rod, hardly-
distinguishable microscopically from the Thiobacillus thioparus, using
the following medium:

Na 2 S 2 3 -5H 2 5.0 grams Agar 20.0 grams

K2HPO4 0.1 gram Tap water 1000 cc.

NaHCOs 0.2 gram

On the plate, both organisms lose their ability to grow very rapidly,
long before they are dead.

The denitrifying organism was studied in greater detail by Lieske, 64
who used a medium having the following composition:

Na 2 S 2 3 -5H 2 5.0 grams MgCl 2 O.lgram

KNO3 5.0 grams CaCl 2 Trace

NaHCOs 1.0 gram FeCl 3 Trace

K 2 HP0 4 0.2 gram Distilled water 1000 cc.

This medium is placed in tall glass cylinders and is inoculated with river
mud containing H 2 S. In a few days, one or more opalescent zones are
formed in the liquid, at some distance from the surface, containing the
denitrifying bacteria in question. The culture is then transferred into an
Erlenmeyer flask filled with the medium and stoppered with a rubber
stopper through which a bent glass tube is passed, one end of which is
dipped in mercury and the rest filled with medium. The culture is
incubated at 25° to 30°C. and in a few days the active formation of nitro-
gen gas takes place. By inoculating the above medium, to which 1.5
per cent washed agar has been added, pure cultures are obtained. The
organism can also be cultivated in dilute meat extract media to which
thiosulfate has been added.

Thiobacillus denitrificans Beij. as described by Lieske is a small
narrow rod, 1/x long, not producing any spores. It is not injured by sun-
shine or oxygen, although it thrives better in its absence. It is auto-
trophic, but is not injured by organic substances. Various carbonates
and bicarbonates can be used as sources of carbon, but C0 2 cannot be
used because of the injurious effect of the free sulfuric acid formed.
In the presence of nitrates, the following substances can be utilized as
sources of energy: hydrogen sulfide, flowers of sulfur, sodium thio-

64 Lieske, R. Untersuchungen liber die Physiologie denitrifizierender Schwe-
felbakterien. Ber. deut. bot. Gesell. 30: 12-22. 1912.

AUTOTROPHIC BACTERIA 87

sulfate and sodium tetrathionate, which are completely oxidized to
sulfate.

The energy liberated in the process is sufficient both for the reduction
of the nitrate, which is an endothermic phenomenon, and the assimila-
tion of C0 2 from the carbonate and bicarbonate. The reason why the
same organism can oxidize various compounds of sulfur, while other
autotrophic soil bacteria, like those concerned in nitrification, can act
only on one definite compound, is explained by Lieske to be due to the
step-like oxidation of the sulfur compounds. For every 100 gm. of
Na 2 S 2 3 oxidized to sulfate, Lieske found that 1 gm. of carbon is
assimilated.

The Thiobacillus denitrificans is of universal occurrence in various soil
types, the number of bacteria increasing with the increase in the carbon
content of the soil. 65 By adding thiosulfate and bicarbonate to the
soil, intensive nitrate decomposition takes place. The organism oc-
curring in various soils varies in activity, so that the forms from com-
posts, forest soils and peat belong to a group which is four times as
active as the form from cultivated soils. Thiobacillus denitrificans
offers, according to Beijerinck, 66 the natural connecting link between
sulfur oxidizing bacteria and denitrifying bacteria.

Trautwein 67 isolated an organism from the soil which was classified
with Thiobacillus denitrificans Beij.; this organism is 1 to 2 by 0.5^ in
size, motile, can reduce nitrate, but can grow also under aerobic condi-
tions; it grows well on organic media and does not precipitate any
sulfur from thiosulfate. The organism is facultative autotrophic,
since it can obtain its carbon both from C0 2 (with thiosulfate as a
source of energy) and from organic substances (in the absence of thio-
sulfate). It was grown on the following medium:

KNO, l.Ogram NaHC0 3 l.Ogram

(orNH 4 Cl) 0.1 gram MgCl 2 0.1 gram

Na 2 HP0 4 0.2gram Distilled water 1000 cc.

Na 2 S 2 3 -5H 2 2.0 grams

To prepare a solid medium, agar is added to the above solution. As organic
media, ordinary bouillon or nutrient agar can be employed.

65 Gehring, A. Beitrage zur Kenntnis der Physiologie und Verbreitung deni-
trifizierender Thiosulfat Bakterien. Centrbl. Bakt. II, 42: 402^38. 1915.

66 Beijerinck, 1920 (p. 547).
6T Trautwein, K. Beitrag zur Physiologie und Morphologie der Thi

bakterien (Omelianski). Outrbl. Bakt. II, 53: 513-548. 1921. /<\Vj\0

LIBRARY

UJ

/A, v ^

^^^^

88 PRINCIPLES OF SOIL MICROBIOLOGY

The bacterium was found to be related 68 to the fluorescein group in the
Lehmann and Neumann system, especially to the Bad. denitrificans
(Stutzer and Burri) L and N.

According to Klein and Limberger 69 the thionic acid bacteria are
capable of oxidizing all sources of sulfur found in the soil (elementary
sulfur, hydrogen sulfide, other sulfides, sulfites and hydrosulfites) to
sulfate and polythionate. The sulfur can also be utilized in the form
of organic sulfur (cystin, albumin, nuclein, meat extract) and is oxidized,
through the sulfur stage, to sulfate. The organism reduces KN0 3
to nitrite and to ammonia, in the oxidation of sulfur. The claim, how-
ever, that this organism can also oxidize NH 4 C1 to nitrite needs further
confirmation, especially since the cultivation of these organisms in
pure culture may not often be a very easy matter.

In this group of thionic acid bacteria, we have another connecting
link, between autotrophic and heterotrophic nutrition in nature.

Group V. Minute, colorless, aerobic, non-spore and non-thread
forming bacteria, acting primarily on elementary sulfur and oxidizing it
rapidly to sulfuric acid (No. 22, PI. IV).

When sulfur is mixed with soil, it is oxidized slowly at first and then
as the soil becomes acid it oxidizes rapidly. If powdered rock phos-
phate is added to the mixture of soil and sulfur, the rock is transformed
into soluble phosphates by the acid formed from the sulfur. This
process has been utilized 70 in composting sulfur, rock phosphate and
soil in various proportions. A direct correlation was found between
the acid formed, as shown by the increase in the hydrogen-ion con-
centration, and the amount of phosphates going into solution. When
a fresh compost is inoculated with some material from an old compost,
the reaction goes on more rapidly, indicating the biological nature of
the process.

By inoculating a medium free from any organic compounds and car-
bonates and containing sulfur as the only source of energy, in addition
to minerals and tri-calcium phosphate as a neutralizing agent, the

68 Trautwein, K. Die Physiologie und Morphologie der fakultativ auto-
trophen Thionsaurebakterien unter heterotrophen Ernahrungsbedingungen.
Centrbl. Bakt. II, 61: 1-5. 1924.

69 Klein, G., and Limberger, A. Zum Kreislauf des Schwefels im Boden.
Biochem. Ztschr. 143: 473^S3. 1923.

70 Lipman, J. G., McLean, H. C., and Lint, H. C. Sulfur oxidation in soils
and its effect on the availability of mineral phosphates. Soil Sci. 2: 499-538.
1916. McLean, H. C. The oxidation of sulfur by microorganisms and its relation
to the availability of phosphates. Soil Sci. 5: 251-290. 1918.

AUTOTROPHIC BACTERIA 89

growth of a bacterium, capable of oxidizing sulfur to sulfuric acid,
was obtained. 71 The acid produced interacted with the tricalcium
phosphate and transformed it into CaS0 4 and monocalcium phos-
phate and finally into phosphoric acid. The bacterium was, however,
accompanied by other organisms, chiefly mold spores, which persisted
in the medium on repeated transfer. Repeated attempts to grow
the bacterium on agar plates failed. After all the calcium of the
phosphate had been transformed into calcium sulfate, the medium be-
came very acid, as low as pH 0.58.

In order to make use of the fact that the organism can withstand a
high acid concentration, the media were prepared with an initial reac-
tion of pH 2.0. This allowed only the development of the sulfur-
oxidizing organism. The high initial acidity accompanied by the
use of high dilutions of the culture in making the transfers (1 : 100,000),
finally resulted in obtaining the culture pure. This was demonstrated
by the fact that no growth took place when inoculated on bouillon and
other media, favorable for the development of bacteria and fungi, even
after 10 to 14 days incubation. Microscopic examinations also demon-
strated the purity of the culture. This organism was described as
Thiohacillus thiooxidans. 72 It is a small, non-motile organism, 0.75
to 1.0 by 0.5 to 0.75/x, producing cloudiness throughout the medium
but without the formation of any pellicle. The two media best adapted
for the growth of this organism have the following composition :

I II

(NHO2SO4 0.2 gram (NH 4 ) 2 S0 4 0.2 gram

MgS0 4 -7H 2 0.5 gram MgS0 4 -7H 2 0.5 gram

KH 2 P0 4 3.0 grams KH 2 P0 4 1.0 gram

CaCl 2 0.25 gram Re-precipitated

Elementary, pow- Ca 3 (P0 4 ) 2 2.5 grams

dered sulfur 10 grams Sulfur 10.0 grams

Distilled water 1000 cc. H 3 P0 4 ( — ) to adjust reaction to

pH = 3.0

Distilled water 1000 cc.

The sulfur in both media and the Ca 3 (P0 4 ) 2 in medium II are weighed out sepa-
rately in the individual flasks into which the media are distributed (100-cc. por-
tions are usually placed in 250-cc. flasks). The media are sterilized for thirty
minutes, in flowing steam, on three consecutive days.

71 Lipman, J. G., Waksman, S. A., and Joffe, J. 8. The oxidation of sulfur by
soil microorganisms. Soil Sci. 12: 475-489. 1921.

72 Waksman, S. A., and Joffe, J. S. Thiohacillus thiooxidans, a new sulfur-
oxidizing organism, isolated from the soil. Jour. Bact. 7: 239-256. 1922.

90 PRINCIPLES OF SOIL MICROBIOLOGY

When a flask with one of these two media is inoculated from a fresh vigorous
culture (seven to fourten days old) of the Th. thiooxidans, growth will be mani-
fested by a uniform turbidity, without any pellicle formation, within four to five
days, at 25 to 30°C, the culture becoming very turbid in seven to eight days;
the sulfur which has been floating on the surface begins to drop down. The same
phenomenon is observed when the medium is inoculated with a little soil con-
taining the bacterium, only the length of time required for development is some-
times a little longer, depending upon the abundance of the organism in the soil,
condition of soil, etc. By using the dilution method, even the approximate
number of the organisms in the soil can be estimated. The culture obtained on
the two media is practically pure, due to the fact that very few other organisms
would develop under these conditions.

The bacterium is strictly aerobic and is benefited both by aeration and
greater surface exposure. When a particle of sulfur from the flask is
examined, it is found to be surrounded by the bacteria. At the same
time there is an intense increase in acidity of the medium.

In the presence of calcium phosphate or carbonate, the sulfuric acid,
as soon as formed, interacts with the calcium salt giving crystals of
CaS0 4 -2H 2 0, which are seen in the culture hanging down from the
particles of sulfur floating on the surface, till finally the bottom of the
flask is covered with gypsum crystals. The organism forms no
spores and is destroyed at 55 to 60°C. in several minutes. The limiting
alkaline reaction is about pH 6.0, which is distinctly acid, while at the
other extreme it will grow at pH 1.0. The optimum lies at pH 2.0 to
4.0. It is possible, however, to accustom the organism to a neutral
and even an alkaline reaction, especially when transferred from one soil
to another before the reaction becomes too acid.

The organism derives its carbon from the C0 2 of the atmosphere;
carbonates and bicarbonates affect it injuriously in so far as they tend
to make the reaction alkaline. The presence of organic substances is
not injurious. As a matter of fact, sugars, like lactose and galactose,
ethyl alcohol, and glycerol may even slightly stimulate growth but
without affecting sulfur oxidation and carbon assimilation. For
establishing the purity of the culture, the organism can be grown on a
solid medium having the following composition: 73

Na 2 S 2 3 -5H 2 5.0 grams CaCl 2 0.25 gram

KH 2 P0 4 3.0grams Agar 20.0 grams

NH 4 C1 0.1 gram Distilled water 1000 cc.

MgCl 2 0.1 gram

71 Waksman, S. A. Microorganisms concerned in the oxidation of sulfur in the
soil. IV. A solid medium for the isolation and cultivation of Thiobacillus thio-
oxidans. Jour. Bact. 7: 605-608. 1922.

AUTOTROPHIC BACTERIA 91

The medium is prepared as usual and sterilized at 15 pounds pressure for
fifteen minutes. Plates and slants are inoculated from a vigorous liquid culture
and incubated at 25° to 30°C. Growth appears in five to six days in the form of
minute straw yellow to cream-colored colonies. Under the microscope, each
colony is found to be surrounded with crystals of gypsum due to the action
of the sulfuric acid, formed from the oxidation of the thiosulfate, upon the
CaCl 2 . This phenomenon is particularly prominent in media containing tri-
calcium phosphate in place of the chloride; a clear zone is formed around each
colony, due to the disappearance of the insoluble calcium salt. 74

When, instead of an ordinary neutral or acid soil, an alkaline soil is
used for composting, the sulfur is also oxidized to sulfuric acid with
the result that the alkalinity of the soil is decreased. When enough
sulfur is added, black alkali soil having a reaction of pH 9.8 can be
made neutral and even acid. At first it was thought that Thiobacillus
thiooxidans is responsible for this oxidation, particularly since the same
acid composts were employed. It is possible, however, that the or-
ganism concerned in the oxidation of elementary sulfur in alkali soil is
of an entirely different nature, approaching more the Th. thioparus of
Beijerinck, in its cultural and some physiological characters, rather than
the Th. thiooxidans. The latter, however, is also found in alkaline
composts, and it is possible that both organisms take an active part in
the oxidation of the sulfur under alkaline conditions. The Th. thioparus
group has its optimum on the alkaline side (pH 7.0 to 9.0), as shown by
Trautwein, while the Th. thiooxidans has its optimum on the acid side.
Both organisms may, therefore, act upon the sulfur under alkaline con-
ditions. However, the phylogenetic relationship of the various species
of Thiobacillus, as well as the differences in their chemical action still
remain to be investigated.

Oxidation of selenium and its compounds. Brenner 75 isolated from the
soil an organism (Micrococcus selenicus, less than 0.5/z in size), which is
capable of oxidizing selenides and using the energy obtained for its
activities. Sodium selenite, sodium thiosulfate or sodium selenate, as
well as litmus, methylene blue or indigo carmin, can be used as hydro-
gen acceptors (or sources of oxygen) but not nitrates, sulfates, sulfites
or tellurites. In addition to selenide, the organism can also use various

74 The occurrence of the Thiobacillus group in the soil has been further studied
by Brown, H. D. Sulfofication in pure and mixed cultures, with special refer-
ence to sulfate production, hydrogen-ion concentration and nitrification. Jour.
Amer. Soc. Agron. 15: 350-382. 1923.

76 Brenner, W. Ziichtungsversuche einiger in Schlamm lebenden Bakterien
auf selenhaltigem Nahrbodem. Jahrb. wiss. Bot. 57: 95-127. 1916.

92 PRINCIPLES OF SOIL MICROBIOLOGY

alcohols (ethyl-, iso-butyl) as well as asparagine and glucose as sources of
energy, but not proteins. The carbon is obtained, however, only from
organic substances (ethyl alcohol). The possible oxidation of elemen-
tary selenium, in the absence of organic matter, with an increase of
acidity of the medium has also been suggested. 76 The Th. thioparus and
Th. thiooxidans were found to be inactive in both cases. The relation
between the oxidation of selenium and of selenide and autotrophic proc-
esses is unknown.

Bacteria oxidizing iron compounds. The nitrifying bacteria are found
to be strictly autotrophic and, in the case of the nitrite formers, are even
injured by the presence of organic matter. The sulfur bacteria are
not injured by the organic matter present in the medium (except Th.
thiooxidans which is injured by nitrogenous substances above 0.2 per
cent) and some of them can even utilize organic materials. The
utilization of complex organic substances is true even to a greater extent
in the case of iron bacteria, only one or two forms of which are known
to require iron compounds as a source of energy, while the majority are
able to derive their energy heterotrophically. The strictly iron bacteria,
or those organisms that are capable of oxidizing ferrous to ferric iron,
whereby the energy obtained is used for the chemosynthetic assimila-
tion of carbon, should be distinguished from those organisms that can
absorb or accumulate iron, when living in media containing iron.
Unlike the latter process the precipitation of iron by true iron bac-
teria is a direct result of utilization of energy from the oxidation of
iron. 77

As early as 1836, Ehrenberg, 78 found that microorganisms play an
important part in the formation of ochraceous deposits of bog iron ore.
The iron precipitating organisms are present universally in nature,
wherever iron-bearing waters occur. They belong chiefly to the thread-
forming bacteria, although a number of them have also been found to
belong to the Eubacteria. Here again we find a similarity between
the iron and sulfur bacteria, a large number of forms belonging to
distinctly different morphological groups. The majority of these forms
belong to the higher bacteria, according to the following classification 88 :

76 Lipman, J. G., and Waksman, S. A. The oxidation of selenium by a new
group of autotrophic microorganisms. Science, N. S. 57: 58. 1923.

77 Winogradsky, 1922 (p. 61).

78 Ehrenberg, C. G. Vorlaufige Mittheilungen fiber das wirkliche Vorkommen
fossiler Infusorien, und ihre grosse Verbreitung. Poggendoff's Annalen 38:
213-227. 1836.

AUTOTROPHIC BACTERIA 93

I. Thread-forming bacteria consisting of sheaths with included cells generally
plainly visible. Reproduction by internally produced conidia or by the separa-
tion of motile and non-motile cells (Trichohacteria).

Crenothrix
Leptothrix
L. ochracea
L. Irichogenes

II. True bacteria:

Gallionella (^Spirophyllum)

Siderocapsa

Sideromonas

Only a few of the numerous forms described are true iron bacteria.

It was assumed that the higher bacteria present a variety of forms,
such as single threads composed of cylindrical cells placed end to end
and generally inclosed in sheaths; ribbon forms, twisted spirally;
cylindrical threads showing false branching, or coiled threads and
ribbon forms produced by the bending of the filaments in the middle
and the twisting of the ends around each other like a rope. 79 Gallio-
nella (Spirophyllum) 80 was considered to be the most abundant of the
iron bacteria. The flat ribbon-like or tape-like threads are twisted in
the form of spirals. These spiral bands may occur as single filaments or
may be coiled together.

Some of the iron bacteria are also capable of oxidizing manganese
salts and precipitate manganese hydrates in their cells; 81 Winogradsky
suggested that we may be dealing here with organisms less specialized
than the other autotrophic bacteria, some being iron-bacteria in the
proper sense (Gallionella, Spirophyllum, etc.), some iron-manganese
bacteria (Crenothrix, Leptothrix, etc.) and some may possibly be
obligate manganese-bacteria.

Winogradsky 82 found in 1888 that Leptothrix will live and grow only
in solutions in which iron is present in the ferrous form; where the living
cells are present, a brown coloration of the sheath takes place due to the
oxidation of the iron salt. The oxidation of ferrous compounds (FeC0 3 )

79 Harder, E. C. Iron depositing bacteria and their geologic relations. Prof,
paper 113, U. S. Geological Survey, Dept. of Interior, 1919.

80 Ellis, D. On the discovery of a new genus of thread bacteria (Spirophyllmn
Jerrugineum Ellis). Proc. Roy. Soc. Edinburgh, 27: 21-34. 1907. Lieske, 1911

(p. 95).

81 Schorler, B. Beitriige zur Kenntnis der Eisenbakterien. Centrbl. Bakt. II,
12: 681-695. 1904; Die Rostbildung in den Wasserleitungsrohren. Ibid. 15:
564-568. 1906.

82 Winogradsky, S. liber Eisenbakterien. Bot. Ztg. 46: 262-270. 1888.

94 PRINCIPLES OF SOIL MICROBIOLOGY

to ferric hydroxide is necessary for the life and growth of the organisms,
this process furnishing the necessary energy to the cell for the assimila-
tion of carbon. According to Molisch, 83 however, Leptothrix will grow
well in iron-free media, particularly in peptone solutions, forming per-
fectly colorless sheaths. If iron or manganese compounds are present
in the solutions, they are oxidized and taken up by the sheaths; even
dead cells (killed by boiling) are able to take up ferric hydroxide in their
sheaths. He, therefore, concluded that the process is merely physico-
chemical and the change of ferrous to ferric compounds is due to simple
chemical oxidation and is not connected with the life processes of the
cell. Similar observations were made by Ellis 84 and others, 85 who
claimed that these bacteria living in iron waters have the power of
attracting ferric hydroxide, which is found in quantity in such waters,
the deposition of iron being merely a purely mechanical process. This
was entirely due to the lack of proper differentiation between the
physico-chemical absorption and chemico-biological oxidation of iron,
on the one hand, and between the obligate and facultative autotrophy,
on the other hand. This led to the general terminology of bacteria
which accumulate iron as "iron bacteria," 86 and frequently also to
absurdities, as in the case of isolation of a bacterium which can ac-
cumulate both iron and calcium (incrustations), where it has been
suggested that iron can be replaced by calcium, although evidently no
oxidation of calcium is possible. 87 It has, however, recently, been sug-
gested 88 that the cells of Gallionella (Spirophyllum) are nothing more than
a product of secretion of a small bacterium, 1.2 by 0.5/jl, consisting of
two coccus-like cells (0.6 by 0.5/x); the threads themselves consist only
of ferric hydrate which dissolves completely in hydrochloric acid, leaving
the living cells only at the end of the threads.

83 Molisch, H. Die Eisenbakterien. Jena. 1910.

84 Ellis, D. A contribution to the knowledge of thread bacteria. Centrbl.
Bakt. II, 19: 507-518. 1907; 26: 321-329. 1910; 31: 499-504. 1911; Iron bac-
teria. Science Progr. 10: 374-392. 1916; Iron bacteria. Methuen & Co., London.
1919.

86 Rullman, W. Uber Eisenbakterien. Centrbl. Bakt. II, 33: 277-289. 1912.
Zikes, H. Vergleichende Untersuchungen iiber Sphaerotilus natans (Kiitzing)
und Cladothrix dichotoma (Cohn) auf Grand von Reinkulturen. Centrbl. Bakt.
II, 43: 529-552. 1915.

86 Lohnis, 1910, p. 704 (p. 28).

87 Brusoff, A. Ferribacterium duplex, eine stiibchenformige Eisenbakterie.
Centrbl. Bakt. II, 45: 547-554. 1916; also 48: 193-210. 1918.

88 Cholodny, N. Die Eisenbakterien. Beitriige zu einer Monographie. G.
Fischer. Jena. 1926.

AUTOTROPHIC BACTERIA 95

Lieske 89 working with Spirophyllum confirmed the original observa-
tions of Winogradsky. The oxidation of FeC0 3 takes place according
to the following equation:

2FeC0 3 + H 2 = Fe 2 (OH) 6 + 2C0 2 + 29 calories
Lieske used a medium having the following composition:

(NH 4 ) 2 S0 4 1.5 grams K 2 HP0 4 0.05 gram

KC1 0.05 gram Ca(N0 3 ) 2 0.01 gram

MgS0 4 0.05 gram Distilled water 1000 cc.

The medium was placed in Erlenmeyer flasks (100 cc.) to a height of 2 cm.,
sterilized and allowed to stand two days. Coarse iron filings, which had been
dry sterilized for one hour at 160°C. were then added, 0.05 gram to each flask.
The flasks were inoculated with a small amount of culture of iron bacteria, placed
under a bell-jar in a cool place, and C0 2 added up to 1 per cent. This resulted in
the formation of about 0.01 per cent of FeC0 3 , which remained constant as long
as there was metallic iron present.

Spirophyllum developed in four days in the cultures to which iron was
added. Pure cultures were obtained by repeated transfers to sterile
flasks. Similar results were obtained in 1888 by Winogradsky for
Leptothrix ochracea, although he did not work with pure cultures.
Organic matter in concentrations of over 0.01 per cent (peptone, aspara-
gine, sugar) produced an injurious effect upon the growth of Spiro-
phyllum. This organism can oxidize no other iron salt, except the
bicarbonate, and also not MnC0 3 , showing it to be strictly auto-
trophic and highly specialized. Leptothrix ochracea was found by
Lieske to be able to utilize manganese carbonate as well as iron car-
bonate as a source of energy and to be facultative autotrophic, capable
of existing also in organic media.

The two media used by Lieske for pure culture study have the following com-
position:

I II

Distilled water 1000 cc. Manganese car-

Agar 10.0 grams bonate saturated

Manganese acetate. . . 0.1 gram solution 1:10

NaHCOs 0.001 per cent

(NH 4 ) 2 S0 4 0.001 per cent

K 2 HP0 4 and
MgS0 4 Traces

One may well agree with Harder 79 that certain iron-depositing organ-
isms, such as Spirophyllum, require ferrous bicarbonate in solution and

89 Lieske, R. Beitrage zur Kenntnis der Physiologie von Spirophyllum ferru-
gineum Ellis, einem typischen Eisenbakterium. Jahrb. wiss. Bot. 49: 91-127.
1911; Zur Ernahrungsphysiologie der Eisenbakterien. Centrbl. Bakt. II, 49:
413-425. 1919.

96 PRINCIPLES OF SOIL MICROBIOLOGY

cannot live without it (obligate autotrophic), others, like Leptothrix,
can live without any iron compounds, but, if they are present, can use
either ferrous bicarbonate (or manganese bicarbonate) or soluble
organic compounds (facultative autotrophic). Others, such as the
various lower bacteria, will use the organic radical of certain soluble
organic iron salts when present but cannot utilize any inorganic iron
salts; in other words, the accumulation or incrustation of iron is purely
mechanical and these bacteria should not be considered as iron bacteria
at all. Among the latter organisms we would probably include the
form (similar to Bac. subtilis), 90 which precipitates ferric hydroxide from
solutions of iron salts, then reduces the hydroxide anaerobically to bog
iron.

Bacteria obtaining their energy from the oxidation of simple carbon
compounds. Methane bacteria. Methane may be produced in appre-
ciable amounts in volcanic eruptions, around oil mines and as a result of
different chemical processes. It is also produced in the anaerobic
decomposition of cellulose, of other carbohydrates, organic acids and
proteins. 91 Swamps, 92 manure heaps and low-lying meadows also
contribute large amounts of methane to the atmosphere.

Although the chemical oxidation of methane has been demonstrated
in various instances, it is primarily a phenomenon accomplished by
microorganisms. According to Harrison and Aiyer, 92 the soil film con-
tains bacteria and algae capable of oxidizing methane and hydrogen and
assimilating methane and C0 2 , increasing the oxygen output. B.
methanicus (Methanomonas methanica Orla-Jensen) was isolated from
the soil by Sohngen. 93 It was found to be a short, motile rod, 2 to
3 by 1.5 to 2fx in size, and could transform methane partly into organic
compounds and partly into C0 2 . In older cultures the organism
became nearly spherical.

90 Mumford, E. M. A new iron bacterium. Jour. Chem. Soc. 103: 645-650.
1913.

91 Omeliansky, W. De la mise en liberte tie methane au cours des processus
biologiques naturels. Arch. Sci. Biol. St. Petersbourg, 12: No. 2. 1906.

92 Harrison, W. H., and Aiyer, P. A. S. The gases of swamp rice soils. II,
Their utilization for the aeration of the roots of the crop. Mem. Dept. Agr.
India, Chem. Ser. 4: 1-18. 1914.

93 Sohngen, N. L. tJber Bakterien, welche Methan als Kohlenstoffnahrung
und Energiequelle gebrauchen. Centrbl. Bakt. II, 15: 513-517. 1906.

AUTOTROPHIC BACTERIA 97

The medium used by Sohngen consisted of:

Distilled water 1000 cc.

K0HPO4 0.05 gram

MgNH 4 PO i -6H 2 0.1 gram

CaS0 4 0.01 gram

Inoculation was made with soil. The atmosphere consisted of one part CH 4

and two parts air. 94

Certain other common bacteria are capable of oxidizing methane, as
in the case of Bad. pyocyaneum 95 and Bad. fluorescens liquefaciens. 96
Miinz 97 isolated a methane oxidizing organism, not identical with that
of Sohngen, which he also called Methanomonas methanica. It thrives
at 18° to 40° with an optimum at 34°C. This organism measured 0.9 to
2.2 by 0.3 to 0.4/x in size, was elliptical to cylindrical, non-motile. High
methane and low oxygen content of atmosphere were best for its
growth, although the organism was aerobic. Hydrogen and carbon
monoxide could not replace methane, although alcohols, carbohydrates
and salts of organic acids could. Nitrogen was utilized both in in-
organic and organic forms. The organism may be considered as
facultative autotrophic although the autotrophy of this organism is
still questionable.

B. hexacarbovorum was found 98 to be able to utilize methane, toluol,
xylol and illuminating gas as the only sources of carbon. Various other
hydrocarbons can also be utilized as sources of energy by bacteria. 99
In this connection mention should be made of the work of Sohngen on
the Mycobacteria {M. ladicola, M. phlei), which were found capable of
deriving their energy from the oxidation of benzol, paraffin, petroleum,
and assimilating the C0 2 of the atmosphere. In regard to the oxidation

. 94 Kaserer, H. liber die Oxydation des Wasserstoffes und des Methans durch
Mikroorganismen. Centrbl. Bakt. II, 15: 573-576. 1906.

95 Sohngen, N. L. Het onstaan en verdwijnen van Waterstof en Methaan
onder den invloed van het organische leven. Proefschrift. Delft. 1906 (Bot.
Centrbl. 105: 371-372. 1907.)

96 Aiyer, P. A. S. The gases of swamp rice soils. V. A methane-oxidizing
bacterium from rice soils. Mem. Dept. Agr. India, Chem. Ser. 5: 177 180. 1920.

97 Miinz, E. Zur Physiologie der Methanbakterien. Diss. Halle, 1915.
98 Stormer, 1907 (p. 47).

99 Tausz, J., and Peter, M. Neue Methode der Kohlenwasserstoffanalyse mit
Hilfe von Bakterien. Centrbl. Bakt. II, 49: 497-554. 1920.

98 PRINCIPLES OF SOIL MICROBIOLOGY

of pure carbon by bacteria, certain investigations 100 point to positive
results and others 101 to negative results.

Bacteria oxidizing carbon monoxide. Carbon monoxide is produced,
in large amounts, in the incomplete combustion of carbon compounds
and, in small amounts, in the decomposition of manure. 102 It can be
oxidized, not only chemically, but also by microorganisms. B. obligo-
carbophilus (Carboxydomonas oligocarbophila Orla-Jensen), isolated
from the soil, could purify the laboratory air rich in CO. 103 The organ-
ism was cultivated on simple inorganic media free from any other carbon
sources. It was found to be a small rod (0.7 to 1.0 by 0.5/x), non-
motile, the cells being united into irregular masses by a slimy substance.
The CO is utilized as a source of energy and is oxidized to C0 2 . 104
It is interesting to note that the organism developing under these condi-
tions was later found to be an actinomyces. 105

Bacteria oxidizing hydrogen. Hydrogen can be oxidized both aero-
bically and anaerobically. De Saussure 106 demonstrated in 1838 that
moist soil will transform hydrogen readily into water, while soil heated
or treated with antiseptic substances is unable to do so. A number of
bacteria were isolated 104 - 107 from soils which were able to oxidize hy-
drogen autotrophically with the formation of water. The organism
isolated from the soil by Kaserer {Hydrogenomonas pantotropha (Kaserer)
Orla Jensen) was an aerobic, short, motile rod, 1.2 to 1.5 by 0.4 to 0.5^
in size, occurring singly or in chains, encapsulated. It was motile by

100 Galle, E. Uber Selbstentzi'vndung der Steinkohle. Centrbl. Bakt. II>
28: 461-473. 1910.

101 Schroeder, H. The bacterial content of coal. Centrbl. Bakt. II, 41:
460^69. 1914.

102 Lohnis, 1910, p. 542.

103 Beijerinck, M. W., and Van Delden, A. tjber eine farblose Bakterie, deren
Kohlenstoffnahrung aus der atmospharischen Luft herrnhrt. Centrbl. Bakt. II,
10: 33-47. 1903.

104 Kaserer, H. Die Oxydation des Wasserstoffes durch (Mikroorganismen.
Centrbl. Bakt. II, 16: 681-696, 769-775. 1906.

108 Lantzsch, 1923 (p. 298).

106 de Saussure, Th. Action de la fermentation sur le melange des gaz oxygene
et hydrogene. Mem. Soc. phys. Hist. Nat. Geneve, 8: 163-190. 1839.

107 Niklewski, B. Ein Beitrag zur Kenntnis wasserstoffoxydierenden Mikro-
organismen. Centrbl. Bakt. II, 20: 469-473. 1908; Niklewski, B. tlber die
Wasserstoffoxydation durch Mikroorganismen. Jahrb. Wiss. Bot. 48: 113-142.
1910. Nabokich, A. J., and Lebedeff , A. F. t)ber die Oxydation des Wasserstoffes
durch Bakterien. Centrbl. Bakt. II, 17: 350-355. 1906; Biochem. Ztschr. 7:
1-10. 1908.

AUTOTROPHIC BACTERIA 99

means of a single polar flagellum. The gelatin colonies were yellow,
smooth, rarely greenish; the gelatin was not liquefied. Yellow to
greenish growth on agar.

Kaserer suggested that both the methane and hydrogen oxidation
phenomena are of great importance in the soil, due to the fact that
these substances, which are produced in the subsoil by anaerobic proc-
esses, are thus oxidized and made available to the soil.

Kaserer's medium consisted of:

K2HPO4 0.5gram NaHC0 3 0.5 gram

MgS0 4 0.2 gram FeCl 3 Trace

NH4CI 1.0 gram Water 1000 cc.

The organism growing on this medium developed poorly under auto-
trophic conditions, the oxidation of hydrogen becoming prominent
in the presence of small amounts of soluble organic matter. 108 A non-
motile bacterium, 1.4 by 0.5/x in size, was isolated, different from the H
■pantotropha of Kaserer. The greatest amount of hydrogen was oxidized
in the presence of 0.01 to 0.03 per cent peptone, nutrose or sodium
asparaginate. In association with certain bacteria, the organism was
much more active.

Niklewski used a medium containing:

NH4CI l.Ogram NaCl 0.2 gram

KH 2 P0 4 l.Ogram FeCl 3 0.0001 gram

MgS0 4 -7H 2 0.2 gram Agar 15.0 grams

NaHCOj l.Ogram Water 1000 cc.

The cultures were placed in a bell-jar, through which purified hydrogen was
passed, at 38° to 35°C. The cultures developed in 3 to 4 days. Two organisms
were isolated:

Hydrogenomonas vilrea formed a pellicle on the surface of the liquid medium.
Small yellow subsurface colonies were formed on the agar. On the surface the
colonies were transparent, folded. The cells are 2/i long. Obligate autotrophic.
No motility observed.

Hydrogenomonas flava formed shining yellow colonies on the surface of the

agar, not spreading as rapidly as the H. vitrea, surface smooth, edge entire;

microscopically, the cells were found to be somewhat smaller (1.5/* long). No

pellicle formation on liquid media. Obligate autotrophic. No motility observed.

By further study, Niklewski 109 isolated an organism (H. agilis) which can oxi-

108 Harrison, W. H., and Aiyer, P. A. S. The gases of swamp rice soils. III.
A hydrogen-oxidizing bacterium from these soils. Mem. Dept. Agr. India, Chem.
Ser. 135-148. 1916.

109 Niklewski, B. Uber die Wasserstoffaktivierung durch Bakterien unter
besonderer Beriicksichtigung der neuen Gattung Hydrogenomonas agilis. Kos-
mos, Lemberg. 1923. (Centrbl. Bakt. II, 40: 430-433. 1914.)

100

PRINCIPLES OF SOIL MICROBIOLOGY

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AUTOTROPHIC BACTERIA 101

dize hydrogen by using the oxygen obtained from the reduction of nitrates and
sulfates and in some cases of citrate, tartrate and oxalate. The presence of
nitrate enables the organism to oxidize hydrogen anaerobically. The organism
can also exist aerobically using free oxygen for the autotrophic oxidation of
hydrogen. It can also exist heterotrophically similar to the other organisms.

For the isolation of pure cultures of hydrogen oxidizing bacteria, Lebedeff 110
used a medium consisting of 1000 parts of water, 2.0 KN0 3 , 0.5 NaH 2 P0 4 , 0.2
MgS0 4 , traces of FeCl 3 and an atmosphere of hydrogen containing 5 to 15 per
cent C0 2 . One hundred cubic centimeter portions of medium were placed in
side-arm flasks, inoculated with soil; after evacuating and introducing the gas,
the flasks were sealed. After 5 to 6 days of growth the organism appeared in the
form of a surface pellicle. After 3 to 4 transfers, the organism was isolated on a
silica gel plate, kept in an atmosphere of hydrogen containing 5 to 10 per cent
C0 2 . After 8 to 10 days, milky-white regular colonies appeared on the plate.
These consisted of rod-shaped (spore forming ?) bacteria, 1.2 to 1.5ju long, motile
by means of a single flagellum. The organism was heterotrophic, forming on
gelatin milky-white colonies which changed later to brownish, and was named
Bac. hydrogenes. Gelatin was liquefied, various sugars, organic acids and pro-
tein derivatives were utilized as sources of carbon. Optimum temperature 26°C.

Recent studies 111 ■ 112 have shown that a number of different species of
hydrogen bacteria are present in the soil. They live autotrophically
with hydrogen as a source of energy and heterotrophically in the
absence of hydrogen, thus being facultative autotrophic. The different
species differ in their sensitiveness to oxygen pressure or in the ability
to use combined oxygen for the oxidation of hydrogen. A newly found
species Bacillus yycnoticus was studied in detail. It is a rod-shaped
organism, 1.5 to 4 by 1.0/*, every preparation containing non-motile
and motile cells, with peritrichic flagellation. In addition to the rods,
true cocci as well as giant cells and thick-walled, egg-shaped cells, ten
times as large as the normal bacterial cell, were found in the culture.
The purity of the culture was established by single-cell isolation, using
Bum's India ink method. The giant cells are involution forms, pro-
duced under special environmental conditions. The spores swell up in
length and width, before they can germinate, and may account for the
egg-shaped figures; they also break up into true cocci.

The organism is grown in an inorganic solution containing sufficient

110 Lebedeff, A. F. Investigations of the chemosynthesis of Bacillus hydro-
genes (Russian). Odessa. 1910.

111 Grohmann, G. Zur Kenntnis Wasserstoff-oxydierender Bakterien. Centrbl.
Rakt. II, 61: 256-271. 1924.

112 Ruhland, W. Beitriige zur Physiologie der Knallgasbakterien. Jahrb.
Wiss. Bot. 63: 321-389. 1924.

102 PRINCIPLES OF SOIL MICROBIOLOGY

iron, the latter being added to the sterilized medium, and with hydrogen
in the atmosphere.

The optimum reaction is at pH 6.8 to 8.7, the limits being pH 5.2 to
9.2. Growth takes place on the surface, at the boundary between the
gas and the liquid; in some cases, the liquid becomes turbid. Partial
pressure of the gases (H 2 and C0 2 ) has an inappreciable influence upon
growth. Respiration of the organism is discussed in detail elsewhere
(p. 403).

M. W. Beijerixck

CHAPTER IV
Bacteria Fixing Atmospheric Nitrogen

Nitrogen fixation in nature. All higher plants, all animals and the
great majority of microorganisms depend for their nutrition on combined
nitrogen, whether organic or inorganic in nature, and can make no
use whatsoever of the great store of gaseous nitrogen in the atmosphere.
Large quantities of nitrogen, therefore, are removed every year from the
soil by the growing crops. In addition to that, several groups of soil
microorganisms are even capable of reducing nitrates and liberate
atmospheric nitrogen. The quantities of manure returned to the soil
are far from sufficient to replace the losses from the soil; the attempt to
replace this loss by artificial fertilizers may be sufficient to supply the
need of the growing plant but not to replenish the losses from the soil.
This is accomplished through the agency of nitrogen-fixing bacteria,
working alone or in symbiosis with higher plants. A small amount of
combined nitrogen is formed by chemical agencies, such as electrical
discharges, and is brought down with the yearly rainfalls, but this hardly
amounts to more than one or two pounds of nitrogen per acre per year,
while ordinary forest trees may remove in the wood and leaves over 50
pounds of nitrogen per acre per year. The rest of the nitrogen is
presumably fixed in the soil by the agency of microorganisms.

The first organisms to be studied in connection with the fixation of
atmospheric nitrogen were the bacteria forming nodules on the roots of
leguminous plants; it was then believed that only those organisms that
live symbiotically on the roots of the plants are able, during this process
of symbiosis, to transform the gaseous nitrogen of the atmosphere
into combined forms. It was later found 1-3 that these bacteria are
also capable of fixing nitrogen in the absence of the host plant, when

1 Beijerinck, M. W. Die Bakterien der Papilionaceenknollchen. Bot. Ztg.
46: 725-735, 741-750, 758-771, 782-790, 797-803. 1888.

2 Maze, M. Fixation de l'azote par le bacille des node-sites des Ldgumineuses.
Ann. Inst. Past. 11: 44-54. 1898.

3 Chester, F. D. Oligonitrophile Bodenbakterien. 4th Ann. Meet. Soc.
Amer. Bact. Washington, D. C. 1902. (Centrbl. Bakt. II, 10: 382. 1903).

103

104 PRINCIPLES OF SOIL MICROBIOLOGY

grown in pure culture. 4 But, in addition to these bacteria, the soil
harbors other organisms, always living non-symbiotically, which are
capable of fixing gaseous nitrogen, in the presence of a proper source of
energy. Berthelot 5 suggested, on the basis of numerous investigations,
that the fixation of atmospheric nitrogen is well distributed among soil
microorganisms. He isolated a number of bacteria from the soil and
found that some of them were able to increase the amount of combined
nitrogen in the medium. Winogradsky 6 demonstrated in 1893 that the
property of nitrogen-fixation is limited to certain specific soil organisms;
the mere growth of an organism on nitrogen-free media was still no
indication that it was capable of obtaining its nitrogen from the gaseous
form of the atmosphere. An organism is considered as unable to fix
nitrogen, unless an actual increase in combined nitrogen has been demon-
strated by chemical analysis. However, Beijerinck 7 found that the
number of bacteria in the soil capable of fixing nitrogen is much larger
than suspected by Winogradsky; he designated as oligonitrophilic those
bacteria that were capable of developing in media containing only traces
of combined nitrogen and considered them as nitrogen-fixing forms.

The first non-symbiotic nitrogen-fixing organism was isolated by
Winogradsky in 1893. Clostridium pastorianum (Bac. amylobacter
A.M. et Bred), an anaerobic organism, was foimd capable of bringing
about an increase in the amount of combined nitrogen in the medium,
in the presence of an available source of energy. Caron 8 soon (1895)
isolated a spore-forming organism, Bac. ellenbachensis a, closely related
to Bac. mycoides and Bac. megatherium, to which he ascribed the prop-
erty of fixing nitrogen. This claim was confirmed by Stoklasa, 9 who

4 Negative results have recently been reported by Barthel, Chr. Meddel.
Centralanst. forsoksv. Jordbr. Bakt. Avd. 43, 1926.

5 Berthelot, M. Fixation de l'azote atmospherique sur la terre vegetale.
Ann. chim. Phys. 13: 5-14, 15-73, 74-78, 78-92, 93-119. 1888. Nouvelles recher-
ches sur les microorganismes du sol fixateurs de l'azote. Bull. Soc. Chim. Ill,
11: 781-783. 1894.

6 Winogradsky, 1893 (p. 107).

7 Beijerinck, M. W. liber oligonitrophile Mikroben. Centrbl. Bakt. II, 7:
561-582. 1901.

8 Caron, A. Landwirtschaftlich-bakteriologische Probleme. Landw. Vers.
Sta. 45: 401-418. 1895.

9 Stoklasa, J. Studien uber die Assimilation elementaren Stickstoffs durch
die Pflanzen. Landw. Jahrb. 24: 827-863. 1893; Biologische Studien uber
"Alinit." Centrbl. Bakt., II, 4: 39, 78, 119, 284, 507, 535. 1898; 5: 350-359.
1899; 7: 257-270. 1901.

BACTERIA FIXING ATMOSPHERIC NITROGEN 105

reported appreciable gains in nitrogen by this organism. 10 The idea
then originated to utilize this organism for soil inoculation and a special
preparation "alinit," in a powdered form, was placed on the market.
This proved to be a failure, but aroused great interest and expectations
among the farmers. Actually this organism was found to be unable to
fix any nitrogen. 11 - 12

The important contribution to the subject of non-symbiotic nitrogen-
fixing bacteria, next to Winogradsky's work, was the isolation of the
aerobic organisms Azotobader chroococcum and Azotobacter agile by
Beijerinck. 13 In addition to the Azotobacter group, Beijerinck and
Van Delden 14 also found that various forms of the genus Granulobacter
(which are actually varieties of Bac. asterosporus (A.M.) Mig.) are
capable of fixing nitrogen. A number of other bacteria, commonly
found in the soil are also able to fix small amounts of nitrogen on arti-
ficial culture media, especially when freshly isolated from the soil.

Algae, fungi and actinomyces do not fix any atmospheric nitrogen.
However, symbiosis between nitrogen fixing bacteria and algae has been
established. 15 - 16 Symbiosis is also probable between bacteria and certain
non-leguminous plants, like Alnus, Eleagnus, Myrica, Coriaria, Ceano-
thus, the bacteria forming nodules on the roots of these plants. Sym-
biosis between bacteria and leaves of certain plants was observed in the
case of Pavetta, 17 Ardisia, 18 Kraussia. 19 Knots are formed at the place of

10 See also Beijerinck, M. W. L'influence des microbes sur la fertilite du sol
et la croissance des vegetaux superieurs. Arch. Neerl. Sci. Exact. Nat., Ser. II,
8: VIII-XXXVI. 1904.

11 Stutzer, A., and Hartleb, R. Untersuchungen fiber das im Alinit enhaltene
Bakterium. Centrbl. Bakt., II, 4: 31-39, 73-77. 1898.

12 Kriiger, W., and Schneidewind, W. Untersuchungen uber Alinit. Landw.
Jahrb. 28: 579-591. 1899.

13 Beijerinck, 1901 (p. 104).

14 Beijerinck, M. W., and Van Delden, A. Uber die Assimilation des freien
Stickstoffs durch Bakterien. Centrbl. Bakt., II, 9: 3-43. 1902.

16 Reinke, J. Symbiose von Volvox und Azotobacter. Ber. deut. bot. Ges.
21: 482-484. 1903.

16 Fischer, H. Uber Symbiose von Azotobacter mit Oscillarien. Centrbl.
Bakt. II, 12: 267-268. 1904.

17 Faber, F. C. Das erbliche Zusammenleben von Bakterien und tropischen
Pflanzen. Jahrb. wiss. Bot. 51: 285-375. 1912; 54: 243-264. 1914.

18 Miehe, H. Die sogenannten Eiweissdrusen an den Blattern von Ardisia
crispa A. D. C. (V. M.). Ber. deut. bot. Ges., 29: 156-157. 1911; 34: 576-580.
1916. Weitere Untersuchungen tiber die Bakteriensymbiose bei Ardisia crispa.
II. Die Pflanze ohne Bakterien. Jahrb. wiss. Bot. 58: 29. 1917.

19 Georgevitch. A new case of symbiosis between a bacillus and a plant.
Bull. Bot. Garden Kew, 1916, p. 105.

106 PRINCIPLES OF SOIL MICROBIOLOGY

penetration of the microbe into the tissues of the plant. According to
Faber, the bacteria bringing about these formations can also fix nitrogen
when not working symbiotically with plants. The bacteria are carried
through the seed, into which they penetrate when the plant is still alive.
The amount of nitrogen thus fixed may be so considerable that in India
Pavetta plants are used as green manure.

Direct nitrogen fixation by higher plants was first suggested by the
early chemists, Priestly and Ingenhouse. Positive results were reported
also by some recent investigators. 20 - 21 However the work of investiga-
tors, like Boussingault, 22 Lawes, Gilbert and Pugh, 23 and the more recent
work of Molliard 24 and numerous others definitely point to the fact that
non-leguminous plants are unable to fix any atmospheric nitrogen.

Classification of nitrogen-fixing bacteria. The nitrogen-fixing bacteria
require organic compounds of carbon for structural and energy purposes.
These organisms can be classified on the basis of their source of carbon,
whether they derive it in a non-symbiotic manner or obtain it from the
growing plant, with which they live symbiotically. None of these
organisms are obligate, since they can also obtain their nitrogen from
organic or inorganic nitrogen compounds.

I. Non-symbiotic nitrogen fixing bacteria

1. Anaerobic bacteria

Bac. amylobacter (Clostridium group, Amylobacter group, Granulo-

bacter group)
Other butyric acid bacteria

2. Aerobic bacteria

(a) Azotobacter group

(b) Radiobacter group

20 Mameli, E., and Pollacci, G. Sur l'assimilatione diretta dell'azoto atmosfer-
ico libero nei vegetali. Atti. 1st. Bot. Pavia, Ser. 2, 15: 159-257. 1911; Centrbl.
Bakt. II, 32: 257. 1912.

21 Lipman, C. B., and Taylor, J. K. Proof of the power of the wheat plant to
fix atmospheric nitrogen. Science, 56: 605-606. 1922; J. Frankl. Inst. 1924, 475-
506.

22 Boussingault, J. B. Recherches sur la vegetation, entreprises dans le
but d'examiner si les plantes fixent dans leur organisme l'azote qui est a l'etat
gazeux dans l'atmosphere. Ann. Chim. Phys. (3) 43: 149-223. 1855.

23 Lawes, J. B., Gilbert, J. H., and Pugh, E. On the sources of the nitrogen of
vegetation, with special reference to the question whether plants assimilate free
or uncombined nitrogen. Phil. Trans. Roy. Soc. London, 151: 431-577. 1861;
Rothamsted Mem. 1, No. 1; 3, No. 1.

24 Molliard, M. L'azote libre et les plantes superieures. Rev. Gen. Bot. 28:
225-250. 1916.

BACTERIA FIXING ATMOSPHERIC NITROGEN 107

(c) Bad. -pneumoniae, Bad. aerogenes, and other non-spore forming

bacteria

(d) Bac. asterosporus group and other spore-forming bacteria.
II. Symbiotic nitrogen-fixing bacteria

1. Bacteria living in the roots of leguminous plants

2. Bacteria living on and in the roots of non-leguminous plants

3. Bacteria living in the leaves of certain plants

Isolation of anaerobic bacteria. For the isolation of bacteria capable
of fixing atmospheric nitrogen, Winogradsky 25 used a solution free from
combined nitrogen of the following composition :

Distilled water 1000 cc. NaCl 0.01 gram

Glucose 20.0 grams FeSO 4 and MnSO 4 . . . Traces

K 2 HP0 4 1.0 gram CaC0 3 30 grams

MgS0 4 -7H 2 0.5 gram

One hundred cubic centimeters of this medium is placed in a flask and 4 grams of
chalk added. The medium is sterilized at 106° to 110° for 30 to 45 minutes. A small
quantity of soil, preferably first pasteurized is used for inoculation. After a few
days' incubation at 25° to 30°, the surface of the liquid becomes covered with a thin
pellicle of aerobic bacteria; gas bubbles are formed abundantly, an indication of
butyric acid fermentation. Gas formation begins from the lump of soil and spreads
all over the flask, so that the whole surface is soon covered with gas, while the chalk
is lumped together by bacterial slime. The culture gives off an odor of butyric
acid and its esters. On examining the culture microscopically, it is found that
the bacteria in the film and in the residue are not alike. The film contains an
aerobic organism, while the residue contains Clostridium {Bac. amylobader) in
abundance, in the characteristic forms. Transfers are made, by inoculating a
piece of chalk from the bottom of the flask into fresh lots of media. When the
culture is sufficiently enriched in Clostridia (after three to four transfers), at-
tempts are made at isolation of pure cultures. Pieces of potato smeared with
chalk are placed in Petri dishes and sterilized. These are inoculated with spore ma-
terial and incubated, under anaerobic conditions, at 30° to 35°, in a partial vacuum
or hydrogen atmosphere. After 5 to 7 days, there appear upon the surface of the
potato elevated, rounded, yellowish colonies filled with gas bubbles. On opening
the apparatus, the colonies can be examined for Clostridia and transfers made
into liquid media or fresh potato cultures. The organism often occurs on the
potato in involution forms, which temporarily lose the capacity of spore forma-
tion. The culture can be grown under aerobic conditions in the presence of an
aerobic non-spore forming organism, such as Bad. fluorescens or Azot. chroococ-

25 Winogradsky, S. Sur l'assimilation de l'azote gazeux de l'atmosphere par
les microbes. Compt. Rend. Acad. Sci. 116: 1385-1388. 1893; 118: 353. 1894;
Recherches sur l'assimilation de l'azote libre de l'atmosphere par les microbes.
Arch. Sci. Biol. (St. Petersburg), 3: 297. 1895; Clostridium pasteurianum, seine
Morphologie und seine Eigenschaften als Buttersaureferment. Centrbl. Bakt. II,
9: 43-62. 1902.

108 PRINCIPLES OF SOIL MICROBIOLOGY

cum. When a pure culture is wanted, the culture is pasteurized (at 75° for 10
minutes), whereby the non-spore forming aerobe is killed and the spore-forming
Clostridium is obtained pure. 26

Bredemann 27 used a solid medium of the following composition:

Glucose 1.0 gram [Agar 1.6 grams

or

Witte peptone 1.2 grams [Gelatin 20.0 grams .

Liebig's meat extract. . 0.8 gram Distilled water 100 cc.

NaCl 0.2 gram Reaction slightly alkaline

The various butyric acid bacteria, including the CI. yastorianum, grow
very well on this medium, which can be used both for the isolation and
cultivation of the organisms.

26 Omeliansky, W. L., and Solounskoff, M. Sur la distribution des bacteries
azotofixatrices dans les sols russes. Arch. Sci. Biol. 18: 1-24. 1915.
"Bredemann, 1909 (p. 109).

PLATE VI
Non-Symbiotic Nitrogen Fixing Bacteria

25. Clostridium pastorianuvi (Bac. amylobacler) , stage preceding spore-forma-
tion; stained with gentian violet, X 660 (from Omeliansky and Solounskoff).

26. Clostridium pastorianum (Bac. amylobacter) , spore-formation, X 660
(after Winogradsky and Omeliansky).

27. Clostridium pastorianum (Bac. amylobacler), large involution forms; dark
color due to coloration of the glycogen with iodine (after Bredemann and
Omeliansky).

28. Azolobacler chroococcum, young culture (from Krzemieniewski).

29. Az. chroococcum, resting forms of sarcina type (from Krzemieniewski).

30. Az. chroococcum, thread forms, individual cells undergoing division (from
Krzemieniewski) .

31. Az. chroococcum, growth on agar, showing the darkening of the culture
(from Krzemieniewski).

32. Az. agile, grown on phosphate-glucose agar, 2 days old, X 660 (from
Beijerinck).

33. Az. agile, showing fiagella, stained by method of Zettnow, X 660 (from
Beijerinck).

34. Bac. asterosporus, 1-5, showing different stages of development and spore
formation; 6, spore with folded envelope; 7, cross section of spore (after Brede-
mann and Omeliansky).

35. Az. vinelandii (from Lipman).

36. Bac. malabarensis: A, culture on meat extract agar; B, on soil infusion-
mannite agar; C, soil infusion mannite solution (from Lohnis and Pillai).

PLATE VI

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BACTERIA FIXING ATMOSPHERIC NITROGEN 109

Morphology of the anaerobic bacteria.

CI. pastorianwn is an actively motile gram-positive rod, occurring singly.
The straight cylindrical rods, with rounded ends, are, when young, 1.5 to2/ilong
by 1.2 to 1.3/u thick. They may reach a size of 2 to 6 by 0.8 to 1.3,u. The proto-
plasm is homogeneous, stains well with aniline dyes and gives a yellow color with
iodine; older cells become spindle-shaped, coloring violet-brown with iodine.
The size of the cells is greatly influenced by the composition of the medium.
Both young and old cells are motile, by peritrichous flagella. When the nutrients
in the medium are exhausted, spore formation takes place; the rods change into
short, thick cells greatly swollen in center, with a diameter twice, or more than
twice as great as the original. The cell membrane becomes sharply contoured
and surrounds an hyaline substance which encloses the spore. The contents
of the cell become granulated, stain with aniline dyes only with difficulty and
color violet with iodine. The spore is formed in one end of the enlarged part of the
cell and stains well with aniline dyes, but not with iodine; methylene blue stains
the spore dark blue and the protoplasm light blue. By the swelling of the hyaline
substance, the mother cell bursts open in one end: the ripe spore, 1.6 by 1.3^, is
now found to lie in a rounded three cornered spore-capsule. It is characteristic
of the species for the capsule to adhere to the spore for a considerable period of
time. Under favorable conditions, as on fresh media, the spores swell up and the
spore envelope breaks. The spore-germination takes place at one pole towards
the open end of the capsule. The young cell soon divides, while the old shell may
remain in the liquid for a long time. The cell also produces various involution
forms, as long threads irregularly swollen and often carrying a spore at one end.
Glycogen and granules accumulate in the cells of the organisms just previous to
spore formation.

Bredemann 28 included all the butyric acid bacteria under the name of
Bac. amylobacter A. M. et Bred., since the various characteristics, such
as size and shape of organism, motility, character of growth on various
media, liquefaction of gelatin, carbon sources, products of metabolism,
deposition of amylaceous material are all variable characteristics. The
most stable characteristics are the form and size of spore as well as the
polar germination. Omeliansky, 29 however, did not agree with the
grouping of all the Clostridia, Granulobacter, Bac. orthobutylicus and
others together, but suggested that some of the characters are sufficiently
constant to be of value in classification.

The nitrogen-fixing capacity is well distributed among the butyric
acid bacteria, including the CI. pastorianum of Winogradsky, CI.

23 Bredemann, G. Untersuchungen fiber die Variation und das Stickstoffbin-
dungsvermogen des Bacillus asterosporus A. M. Centrbl. Bakt. II, 22: 44-89.
1908; 23: 385-568. 1909; Ber. deut. bot. Gesell. 26: 362 and 795. 1908.

29 Omeliansky, W. L. Morphological and cytogical investigations on nitrogen-
fixing bacteria (Russian). Arch. Sci. Biol., 20: 24-49. 1916.

110 PRINCIPLES OF SOIL MICROBIOLOGY

americanum, a facultative aerobic organism, isolated by Pringsheim, 30
Granulobacter pectinovorum of Beijerinck and Van Delden, and others.

Distribution of anaerobic nitrogen-fixing bacteria in the soil. Wino-
gradsky has already demonstrated the wide occurrence of Clostridium,
which he isolated from every soil sample taken in St. Petersburg
and in Paris. A larger form was found in southern Russia. More
recent studies 31 demonstrated the presence of this organism in practically
all the Russian soils; the various strains found in different localities
vary greatly in their morphology. Similar results were obtained for
German soils. 32 An examination of 152 different soil samples taken in
different parts of the world indicated the presence of Bac. amylobacter
in 137 cases, including surface soils and subsoils, cultivated and virgin
soils, except in a few acid peat soils. 33 The occurrence of Bac. amylo-
bacter in Vesuvian soils has also been pointed out. 34

The number of nitrogen-fixing Clostridia in the soil was found to be
over 100,000 per gram, or much more abundantly than Azotobacter. 35
This led various investigators to conclude that the Clostridium rather
than Azotobacter is the most important group of non-symbiotic nitro-
gen-fixing bacteria. Duggeli 36 found 100 to 1,000,000 anaerobic and
to 100,000 aerobic-nitrogen-fixing bacteria per gram of soil. Plots
receiving sodium nitrate as a source of nitrogen contained 10,600 to
12,000 Bac. amylobacter and 4,900 to 6,300 Azotobacter cells; plots re-
ceiving no nitrogen, but potassium and phosphorus-fertilizers, contained
1,120,000 Bac. amylobacter and 98,700 Azotobacter cells per gram of soil.

Physiology of anaerobic nitrogen- fixing bacteria. Winogradsky found
CI. pastorianum to be an obligate anaerobic form which can develop
under aerobic conditions only in the presence of aerobic bacteria. How-
ever, the aerobic CI. americanum isolated by Pringsheim was very similar

30 Pringsheim, H. tlber ein stickstoffassimilierendes Clostridium. Centrbl.
Bakt. II, 16: 795-800. 1906; 20: 248-256. 1907; 21: 673. 1908; 23: 300. 1909;
24:488-496. 1909;36:468-472. 1913;40:21-23. 1914.

31 Omeliansky and Solounskoff, 1915 (p. 108).

32 Freudenreich, E. v. Ueber stickstoffbindende Bakterien. Centrbl. Bakt.
10: 514-522. 1903.

33 Haselhoff, E., and Bredernann, G. TJntersuchungen iiber anaerobe stick-
stoffsammelnde Bakterien. Landw. Jahrb. 35: 381^414. 1906.

34 Riccardo, S. Primo contributo alia conoscenza dei Batteri fissatori di azoto
nei terreni vesuviani. Ann. R. Sc. Sup. Agr. Portici, 18: 1-50. 1923.

36 Truffaut, G., and Bezssonoff, N. Augmentation du nombre des Clostridium
Pastorianum (Winogradsky) dans les terres partiellement sterilisees par le sulfure
de calcium. Compt. Rend. Acad. Sci. 172: 1319-1322. 1921.

36 Duggeli, 1921 (p. 39).

BACTERIA FIXING ATMOSPHERIC NITROGEN 111

in morphology to the other Clostridia, but was capable of fixing larger
quantities of nitrogen, perhaps due to its aerobic nature. Bredemann,
after examining a large number of Clostridia, came to the conclusion
that they are all capable of developing more or less even in the presence
of air and fix nitrogen. The presence of 30 mgm. of oxygen per 1 liter
of air will still allow spore germination.

The optimum temperature for the development of the organism is
28° to 30°C. The spores are not destroyed at 75° even at the end of 15
hours; at 100°C the spores are destroyed in five minutes.

CI. pastorianum utilizes glucose, maltose, lactose, levulose, sucrose,
galactose, maltose, raffinose, dextrin, inulin, glycerol, mannite and
lactates. Winogradsky found that the nitrogen source greatly in-
fluences the nature of the carbon sources that can be utilized. The
greater the concentration of sugar the lower is its economic utilization,
3.2 mgm. nitrogen being fixed per gram of glucose in 0.5 per cent solu-
tion, 2 mgm., in 2 per cent solution and 1.2 mgm. in 4 per cent solution.
In the presence of combined nitrogen, nitrogen fixation decreases and
comes to a standstill, when the solution contains more than six parts of
combined nitrogen in one thousand parts of solution (Winogradsky).
Omeliansky, however, observed some fixation even with a concentration
of 16 parts of combined nitrogen.

The optimum reaction for the growth of CI. pastorianum is pH 6.9
to 7.3, but it still develops well at pH 5.7. It can withstand a greater
acidity than proteolytic anaerobes like Bac. putrificus, from which it can
be thus freed. 37 The addition of CaC0 3 to the glucose medium has a
favorable effect, in neutralizing the acids formed ; MgC0 3 is less favora-
ble. CI. pastorianum can thus withstand a greater acidity than Azoto-
bacter, whose limit is pH 6.0. In acid soils (more acid than pH 6.0)
Azotobacter is inactive while the butyric acid bacteria may still be
abundant.

When freshly isolated, the Clostridium fixes more nitrogen than when
cultivated for a long time in artificial media. The culture can be
invigorated by growing it in Winogradsky's liquid medium, to which
enough ammonium sulfate is added so as to offer the organism less
nitrogen than is needed for the complete decomposition of the sugar.
By transferring from this culture, when gas formation ceases, normal
growth and nitrogen fixation is obtained. Bredemann 38 invigorated the
culture by passing it through soil.

"Dorner, 1924 (p. 165).

38 Bredemann, G. Die Regeneration des Stickstoffbindungsvermogens der
Bakterien. Centrbl. Bakt. II, 23: 41-47, 385-568. 1909.

112 PRINCIPLES OF SOIL MICROBIOLOGY

A fertile garden soil is dried, sieved and placed in a flask to a height of 5 cm.
The soil is moistened with water and sterilized in an autoclave for 45 minutes at
150°C. When the soil is found to be sterile, it is inoculated with an emulsion of a
fresh growth of the weakened culture grown on agar. The flasks are allowed to
incubate, one at room temperature under aerobic conditions and one at 28° under
anaerobic conditions, for one month, then at room temperature under aerobic
conditions. The soil has all dried out by this time. When two grams of it is
inoculated into sterile liquid medium, active growth and gas formation begins
in 12 hours, reaching a maximum in 36 hours.

It was found that this invigorated culture would coagulate milk with
gas formation. Most strains, however, would not grow upon milk. Gel-
atin was not liquified and casein was not decomposed. Ammonia was
not formed from peptone, nitrates were not reduced.

Nun-symbiotic nitrogen-fixing aerobic bacteria. When a simple medium
containing tap water, 0.02 per cent K 2 HP0 4 and glucose as a source of
carbon is inoculated with soil and incubated in the dark, CI. pastoria-
num, together with other bacteria, is obtained. When the glucose is
replaced by mannite (2 per cent) or by propionate of potassium or
sodium, another large organism predominates; Beijerinck called this or-
ganism Azotobacter chroococcum and found it in all soils and manures. 39
On repeated transfer to fresh lots of sterile media, the organism was
gradually purified from the majority of contaminating organisms and
finally isolated on mannite agar. In addition to the above simple
medium several other media are used successfully for the isolation of
Azotobacter:

1. Beijerinck medium:

Tap water 1000 cc.

Mannite 20 grams

K 2 HP0 4 0.2 gram

The mannite can be replaced by dextrin, glycerol, calcium malate (0.5 per cent)
and other salts of organic acids. The water may be replaced by soil extract. 40

2. Lipman's solution: 41

Distilled water 1000 cc. MgS0 4 -7H 2 0.2 gram

Mannite 15 grams CaCl 2 0.02 gram

K2HPO4 0.2 gram FeCl 3 1 drop of 10 per cent solution

39 Beijerinck, 1901 (p. 104).

40 Lohnis, F. Beitriige zur Kenntnis der Stickstoffbakterien. Centrbl. Bakt.
II, 14: 582-604, 713-723. 1905; Landwirtschaftlich-bakteriologisches Praktikum.
1911. P. 131.

41 Lipman, J. G. Further contributions to the physiology and morphology of
members of the Azotobacter group. N. J. Agr. Exp. Sta. 25th Ann. Rpt. 1904,
237-289. Lipman, J. G., and Brown, P. E. A laboratory guide in soil bacteri-
ology. 1911.

BACTERIA FIXING ATMOSPHERIC NITROGEN 113

The substances are dissolved, and enough 10 per cent NaOH is added to make
faintly pink to phenolphthalein.

3. Ashby's solution: 42

Distilled water 1000 cc. NaCl 0.2 gram

Mannite 15 grams CaS0 4 -2H 2 0.1 gram

K2HPO4 0.2gram CaC0 3 5.0 grams

MgS0 4 -7H 2 0.2 gram

Make neutral to phenolphthalein with NaOH solution.

4. Omeliansky's solution: 43

Distilled water 1000 cc. MgS0 4 -7H 2 0.5 gram

Dextrin 20 grams CaC0 3 10.0 grams

K2HPO4 1.0 gram

Solid media are prepared by adding 15 or 20 gm. of agar to the above solutions.

For the study of pigment formation the last medium is very appropriate. Potato

and potato agar, milk, mannite-nitrate media are also employed for the study

of the morphology of the organism.

A small quantity of fertile garden soil, or well limed and manured field
soil is used for the inoculation of the sterile liquid medium. After a few
day's incubation at 25° to 30°C, a pellicle is formed on the surface of the
liquid, at first gray in color, later becoming brownish. A microscopic
examination of the pellicle shows the presence of the typical Azotobacter
cells with large slimy capsules. A part of the pellicle is then transferred
into a fresh flask with sterile medium. After several transfers, the culture
is sufficiently enriched in Azotobacter so that one can proceed to isolate
it on agar media. A loopful of material from a young culture, in
which no heavy pellicle has as yet been formed, is diluted in a few cubic
centimeters of sterile water to separate the Azotobacter cells. A second
and third dilution is made and the surfaces of a series of Petri dishes, in
which liquefied mannite agar has been placed and allowed to cool, are
then streaked out with the suspensions of the various dilutions. At
25°C, pale, rounded, raised colonies are formed in a few days. In addi-
tion to these, transparent raised colonies of Radiobacter are also found.
The Azotobacter colonies are carefully selected by microscopic examina-
tion and are transferred into sterile liquid media. These precautions
are very important so as not to have the culture contaminated.

The silica gel plate, to which minerals and mannite have been added,
inoculated with small particles of soil will give directly a nearly pure
culture of Azotobacter, as shown by Winogradsky. 44

42 Ashby, S. F. Some observations on the assimilation of atmospheric nitrogen
by a free living soil organism, Azotobacter chroococcum of Beijerinck. Jour. Agr.
Sci. 2: 35-51. 1907.

43 Omeliansky, W. L., and Sseverowa, O. P. Die Pigmentbildung in Kulturen
des Azotobacter chroococcum. Centrbl. Bakt. II, 29: 643-650. 1911.

"Winogradsky, 1925 (p. 11).

114 PRINCIPLES OF SOIL MICROBIOLOGY

Description of species of Azotobacter. Azotobacter is a bacterium, 4 to
6^ broad, forming, when young, diplococci or short rods, with hyaline
contents, often containing a vacuole and forming a slimy wall. Various
involution forms, as threads reaching to 60 to 80m in length, may be
formed. Formation of nuclei has been demonstrated by Prazmowski.
The younger cells are motile, by means of the 4 to 10 polar flagella,
which are about as long as the bacteria. The organisms are aerobic,
readily killed on heating. Beijerinck, Lipman and Jones 45 denied the
existence of spores in Azotobacter. According to Ashby, Fischer and
Krzemieniewsky, spore formation is positive, since the organism can
resist drying for a long period of time. Prazmowski 46 demonstrated that
spore formation takes place under normal conditions, with sufficient
aeration and in the presence of humates. The process of spore forma-
tion is later described in detail. 47 - 48

Azotobacter chroococcum Beij. is universally distributed in soils having a reac-
tion above pH 6.0. It produces a crude floating membrane on tap water contain-
ing 2 per cent mannite and 0.02 per cent K2HPO4 inoculated with garden soil-
Only occasional individuals in young cultures are motile by means of a single
flagellum. The size of the cell is differently reported by investigators, varying
from 3 to 4 by 5 to 6/* and even 3 to 4 by 9 to 12)u to 2 to 3 by 3 to 4ju (limits 1.5 to
7n) and even 1 to 2p for cocci and 1.5 to 2 by 3 to 4/* for rods. 49 Old membranes
consist of micrococci of various sizes united to form sarcina-like masses, possessing
mucilaginous walls. The older cultures are frequently brown or black. This
organism oxidizes numerous carbon compounds to C0 2 and H 2 0. According to
Stoklasa, 60 organic acids are also formed; however, Omeliansky suggested that
Stoklasa probably worked with contaminated cultures.

Azotobacter agile Beij. was found universally distributed in canal waters of
Delft; the crude and pure cultures are obtained by previous methods. The

46 Jones, D. H. A morphological and cultural study of some Azotobacter.
Centrbl. Bakt., II, 38: 14-21. 1913. Further studies on the growth cycle of
Azotobacter. Jour. Bact. 5: 325. 1920.

46 Prazmowski, A. Die Entwicklungsgeschichte, Morphologie und Cytologie
des Azotobacter chroococcum Beij. Centrbl. Bakt. II, 33: 292-305. 1912; 37:
299. 1913. Azotobacter-Studien. II. Physiologie und Biologic Ariz. Akad.
Krakau. Math.-Naturw. Kl. (B), 1912, 855-950. (Centrbl. Bakt. II, 37: 299-301.
1913).

47 Lohnis and Smith, 1916 (p. 56).

48 Mulvania. Observations on Azotobacter. Science, 42:463. 1915.

49 Bonazzi, A. Cytological studies of Azotobacter chroococcum. Jour. Agr.
Res., 4: 225-241. 1915.

60 Stoklasa, J. Beitrag zur Kenntnis der chemischen Vorgiinge bei der Assimi-
lation des elementaren Stickstoffs durch Azotobacter und Radiobacter. Centrbl.
Bakt. II, 21: 484-509, 620-632. 1908.

BACTERIA FIXING ATMOSPHERIC NITROGEN 115

organism is very motile by means of a bundle of polar flagella. The cells are
large, transparent, resembling monads, often with a clearly discernible cell wall,
protoplasm, nucleus, granules and vacuoles. In the presence of salts of organic
acids, it produces a green or red diffusible pigment.

Azotobacter vinelandii was isolated by Lipman 51 from a New Jersey soil in 1903.
In four days it forms on mannite agar colonies 4 mm. in diameter. These are
round, raised, concentric and semi-transparent, with denser whitish centers;
the deep colonies are white, small, hardly more than 1 mm. in diameter, elliptical
to spindle-shaped. A white thick membrane is formed on the mannite solution.
When undisturbed, the liquid culture shows the formation of a bright yellow pig-
ment, concentrated near the surface and gradually diffusing through the liquid.
In older cultures the pigment gradually diffuses throughout the medium and
becomes darker, until in old cultures it may become a yellowish red. At the same
time the bacterial mass may also become darker. A considerable number of
forms, ranging from large rods with rounded ends to spherical organisms, are found
in mannite cultures. Most of the organisms are actively motile, showing pro-
gressive and at times rotatory motility. As the culture grows older, the number
of shorter rods increases, and the cells begin to accumulate and store up fat,
which appears in small globules throughout the bacterial body and gives it a
granular appearance. Various involution forms are produced in meat extract
bouillon. A temperature of 85°C. for five minutes is sufficient to destroy all the
cells. The organism stains readily with carbol fuchsin, with aqueous solutions
of gentian violet, methyl violet or fuchsin and Loffler's methylene blue. The
bright yellow pigment produced in mannite solution is favored by greater surface
of medium (oxygen need), is soluble in alcohol and decolorized by weak acids.
The organism is very active in the fixation of atmospheric nitrogen.

Azotobacter beijerinckii was isolated by Lipman in 1904. It forms pure white,
moist, soft, irregularly round, netted colonies. In mannite solution, the organ-
ism forms a turbidity, with white circular dots on surface and walls, gradually
settling to the bottom. The cells are large, almost spherical, occurring singly,
or in chains of two or more. This organism is much larger than A. chroococcum
and A. vinelandii and does not show any motility. On solid media, a yellowish
pigment is formed. Azotobacter vitreum was isolated by Lohnis and Westermann. 82
It forms only round cells, is non-motile, grows as transparent, moist colonies on
solid media, without any pigment. Prazmowski doubts whether this organism
belongs to the genus Azotobacter.

Distribution of Azotobacter in the soil. Azotobacter is of universal
occurrence in the soil, 53 but not to such an extent as Clostridium. Out
of one hundred and five soil samples examined, Burri 54 found Azoto-

51 Lipman, J. G. Experiments on the transformation and fixation of nitrogen
by bacteria. N. J. Agr. Exp. Sta. Rpt. 24: 217-285. 1903. 25th Ann. Rpt. 1904,
237-289; Azotobacter studies. Ibid. 26: 254. 1905; 29: 137. 1908.

62 Lohnis, F., and Westermann, T. Uber stickstofffixierende Bakterien.
Centrbl. Bakt. II, 22: 234-254. 1908.

83 Beijerinck and Van Delden. 1902 (p. 105).

64 Burri, R. Die Nutzbarmachung des LuftstickstofTs durch Bodenbakterien.
Schweiz. Ztschr. Forstwesen. 55: 89. 1904.

116 PRINCIPLES OF SOIL MICROBIOLOGY

bacter missing in thirty-four cases, chiefly in heavy clay soils. Jones
and Murdoch 55 found Azotobacter in nine out of seventeen soil types
examined and in twenty-two out of twenty-nine soil samples repre-
senting nine types. Eighteen was the maximum number of Azotobac-
ter cells found per one gram of soil.

The absence of Azotobacter in the soil is probably due, in the majority
of cases, to the soil reaction, since this organism cannot develop in a soil
having an acidity greater than pH 6.0. As soon as the reaction of the
soil is adjusted by means of lime so that the pH becomes greater than
6.0, an Azotobacter flora will develop. 56-58

Azotobacter is widely represented in the soil by various forms, so
that Lohnis and Westermann counted in 1908 as many as twenty-one
forms. These were believed, however, to represent only four types.
Lipman and Burgess 59 isolated in 1915 a number of forms from Ameri-
can and foreign soil including several new species. The most common
form, however, was in both cases A. chroococcum.

As a result of a study of a large number of soils, Lipman and Burgess 59
came to the conclusion that, with a proper supply of energy producing
materials, all agricultural soils may be made to fix atmospheric nitrogen
when inoculated into a properly constituted mannite solution; however,
only a fraction of these soils (one-third) contain Azotobacter organisms.
Those soils that contain Azotobacter have a more vigorous nitrogen
fixing power. As much as 10 mgm. of nitrogen are fixed per gram of
mannite in solution and 12.6 mgm. in the soil by pure cultures of
Azotobacter, which approaches that obtained from A . vinelandii. The
latter is found to be the strongest nitrogen fixing organism, while A.
chroococcum the most common.

66 Jones, D. H., and Murdoch, F. G. Quantitative and qualitative bacterial
analysis of soil samples taken in the fall of 1918. Soil Sci. 8 : 259-267. 1919.

66 Christensen, H. R. Untersuchungen iiber einige neuere Methoden zur
Bestimmung der Reaktion und des Kalkbediirfnisses des Erdbodens. Intern.
Mitt. Bodenk. 13: H. 3-4. 1923.

57 Gainey, F. L. A study of the effect of changing the absolute reaction of
soil upon their Azotobacter content. Jour. Agr. Res. 24: 289-296, 759-767, 907-
938. 1923.

53 Waksman, S. A. The occurrence of Azotobacter in cranberry soils.
Science, 48: 653. 1918.

69 Lipman, C. B., and Burgess, F. A. Studies on nitrogen fixation and Azoto-
bacter forms in soils of foreign countries. Centrbl. Bakt. II, 44: 481-511. 1915.

BACTERIA FIXING ATMOSPHERIC NITROGEN 117

Azotobacter was found in most Javan soils, 60 in all soils in India 61 in a
half of Polish soils 62 and in about 33 per cent of the cultivated soils in
Japan. 63 The wide distribution of Azotobacter in Russian soils, in
Utah soils 64 and in Danish soils 65 has also been pointed out. How-
ever, Azotobacter is almost completely absent in Finnish soils, even
those that are well buffered and supplied with CaC0 3 . 66

Azotobacter derived from different localities may vary greatly in the
amounts of nitrogen fixed. In humid regions the nitrogen-fixing or-
ganisms are confined to the upper few inches of soil. 67 However, in
arid regions, they may be quite active to a depth of 3 to 4 feet. Azoto-
bacter occurs more frequently in cultivated than in virgin soils. The
number of Azotobacter in the soil is highest in spring and fall of year
and lowest in summer and winter. According to Beijerinck, the num-
ber of Azotobacter in the soil runs parallel with soil fertility. It is
interesting to note that Azotobacter is among the first organisms to
develop in a newly formed soil, as in the case of Vesuvian soils. 68

Lohnis and Smith recognized only two species of Azotobacter isolated
so far: A. cliroococcum and A. agile Beij. (Syn. A. vinelandii J. G.
Lipman). A. beijerinckii Lipman was looked upon as a variety of A.
chroococcum and A . vitreum Lohnis as a variety of A . agile.

Morphology and life cycle of Azotobacter. The form and size of
Azotobacter cells depend upon the composition of the medium and
conditions of cultivation. Increased aeration brings about a lengthen-
ing of the forms and greater motility. In the presence of organic
colloidal substances, especially those containing nitrogen, as well as
aluminum salts, the cells remain in a young condition for a long time;

60 Groenewege, J. Occurrence of Azotobacter in tropical soils. Arch. Sui-
kinderind. 21: 790-793. 1913.

61 Hutchinson, C. M. Soil bacteriology. Rpt. Agr. Res. Inst. Pusa. 1911-12,
85-90; Azotobacter and nitrogen fixation in India soils. Mem. India Agr. Exp.
Sta. Bact. Ser. 1: 98-112. 1915.

62 Ziemiecka, J. Presence de l'azotobacter dans les sols polonais. Rocznikow
Nauk Rolniczych. 10: 1-78. 1923.

63 Yamagata, U., and Itano, A. Physiological study of Azotobacter chroococ-
cum, beijerinckii and vinelandii types. Jour. Bact. 8: 521-531. 1923.

64 Greaves, J. E. Azofication. Soil Sci. 6: 163-217. 1918.

66 Weis, F., and Bornebusch, C. H. On the presence of Azotobacter in Danish
woods. Det. Forstlige Forsogs. Danmark, 4: 319-331. 1914.

66 Brenner, W. Azotobacter in finnlandischen Boden. Geolog. Konn. Fin-
land. Agr. Geol. Meddl.20: 1924.

67 Ashby, 1907 (p. 113).

68 Riccardo, 1923 (p. 110).

118 PRINCIPLES OF SOIL MICROBIOLOGY

alkali salts and other salts stimulate the maturity of the culture.
Prazmowski also observed changes of the dark brown A. chroococcum
into a colorless race similar to A. vinelandii and A. agile and a yellow
race similar to A. beijerinckii; he concluded, therefore, that the various
species described are merely races of one greatly variable species. These
results are confirmed by Omeliansky, 69 who found that pigment forma-
tion by Azotobacter depends entirely on the composition of the medium.
However, this is not sufficient to deny the existence of the various
species altogether, especially since the genus itself is very variable, and
it is difficult to establish the limits for its systematic position. The
organism most commonly studied is A. chroococcum. It goes through a
regular life cycle. Drying stimulates spore formation. The membrane
surrounding the cell becomes more compact and thinner while the cell
itself changes into a spore. 70 Prazmowski observed spore formation
also in ordinary media, containing soil extract. Several spores were
found to be produced in a cell and these are believed to be responsible
for the irregular packet and sarcina forms observed in mature cultures.
The latter are formed by simple fission of the cell. 71 The granules
arising from the splitting up of the supposed nuclear body may act as
gonidia spores.

Lohnis and Smith claim that the genus Azotobacter is characterized
by seven different cell types:

1. Large non-sporulating, globular, oval, or rod-like cells, with polar or

peritrichous flagella.

2. Coccoid cells, the vegetative growth of the regenerative bodies, identical

with Micrococcus.

3. Dwarfed cell type, the vegetative growth of the gonidia.

4. Irregular, fungoid cells, similar to Mycobacterium.

5. Small non-sporulating rods, identical either with Bad. lactis viscosum or

Bad. putidum.

6. Small sporulating rods, identical either with Bac. fusiformus or Bac.

pumillus A. M. et Gotteil.

7. Large sporulating cells.

69 Omeliansky, 1916 (p. 109).

70 Fischer, H. Ein Beitrag zur Kenntnis der Lebensbedingungen von stick-
stoffsammelnden Bakterien. Centrbl. Bakt. II, 14: 33-34. 1905; 15: 235-236.
1906.

71 Jones, D. H. A morphological and cultural study of some Azotobacter.
Centrbl. Bakt. II, 38: 14-25. 1913; 42: 68-9. 1914; Trans. Roy. Soc. Canada.
Ser. 3, 7: 1913, Sect. IV. Further studies on the growth cycle of Azotobacter.
Jour. Bact. 5: 325-342. 1920.

BACTERIA FIXING ATMOSPHERIC NITROGEN 119

The reproductive organs of Azotobacter are :

1. Gonidia, in part filterable, produced in the cell.

2. Regenerative bodies and exospores, either produced by the cells or growing

up from the symplasm.

3. Arthrospores formed by fragmentation of the rod-like or fungoid cells.

4. Mycrocysts, globular or oval resting cells.

5. Endospores, produced singly by the rod-like cells in terminal or in central

position, or in the form of two or more globular or spindle-shaped spo-
rangia.
Gonidia form the basis for the development of regenerative bodies, arthro-
spores and endospores. Symplasm formation and regeneration of new cells
proceeds with Azotobacter as with other bacteria. 72

Physiology of Azotobacter. There is an important difference in the
amount of nitrogen fixed by pure cultures of the organism and by-
crude cultures in the presence of other organisms, in favor of the last.
This was first recognized by Beijerinck and Van Delden, who went as
far as to state that A. chroococcum is incapable of fixing any appreciable
quantities of nitrogen, when growing in pure culture, but large amounts
of nitrogen are fixed in the presence of the spore bearing Granulobacter
or non-spore bearing members of the B. aerogenes group and B. radiobac-
ter. Members of the Granulobacter group were found capable of fixing
nitrogen by themselves, this power becoming very pronounced in the
presence of Azotobacter. On the other hand, Gerlach and Vogel 73
proved conclusively that A . chroococcum is able to fix large quantities of
atmospheric nitrogen when grown in pure culture, in the presence of
salts of organic acids or sugar. This was soon confirmed by others. 74,75
The presence of other organisms, however, is advantageous to the
amount of nitrogen fixed and the rate of fixation, either by using up the
waste products or creating otherwise favorable conditions. Young
cultures will fix more nitrogen than old cultures; crude cultures more
than pure cultures.

A number of hexoses (glucose best), pentoses, alcohols (mannite)
and salts of organic acids, such as malate, 76 lactate, butyrate, succinate,

72 On the systematic position of Azotobacter, the work of Lohnis and Hanzawa
should be consulted. Lohnis, F., and Hanzawa, J. Die Stellung von Azotobacter
im System. Centrbl. Bakt. II, 42: 1-8. 1914.

73 Gerlach, M., and Vogel, I. Stickstoffsammelnde Bakterien. Centrbl.
Bakt. 8: 669-674. 1902; 9: 817-821, 881-892. 1902; 10: 363-643. 1903.

74 Freudenreich, 1903 (p. 113).

75 Lipman, 1903 (p. 115).

76 Beijerinck, M. W. Uber ein Spirillum, welches freien Stickstoff binden
kann? Centrbl. Bakt. II, 63: 353-359. 1925.

120 PRINCIPLES OF SOIL MICROBIOLOGY

can be utilized as sources of energy. The available evidence seems to
indicate that celluloses may be utilized as sources of energy, when
cellulose decomposing bacteria are present, but not directly. 77 Ammo-
nium salts are utilized more readily than nitrates as sources of nitrogen.
Only traces of minerals are required, but the organism can withstand
as much as 10 per cent MgS04. It resists drying very readily and is
sensitive to high temperatures: the cells are destroyed in a few minutes
at 55°C, but resist drying for many years. 78

Other non-symbiotic nitrogen-fixing bacteria. In addition to the CI.
pastorianum and the Azotobacter group, various other bacteria are
capable of fixing appreciable quantities of nitrogen, as pointed out by
Beijerinck and van Delden for a freshly isolated culture of Bacillus
mesentericus, and for a number of species of the aerobic Granulobacter.
The same is true of Bact. lactis viscosum, Bad. pneumoniae, Bad.
radiobader and Bact. prodigiosum?^ Bad. aerogenes, Bact. pyocyaneum
and a number of other bacteria were also found capable of fixing
small amounts of nitrogen, especially when freshly isolated from the
soil. The claims that various spore-forming bacteria, like Bac. sub-
tilis, Bac. megatherium, etc., are also capable of fixing nitrogen were
found to be unfounded. However, two representatives of the Bac.
mesentericus group, namely Bac. malabarensis and Bac. danicus, the
first isolated from South Indian rice soil and the second from
nodules of Vicia, were found capable of fixing nitrogen. Bact. radi-
cicola, the legume organism, can live in the soil outside of the
nodules and may fix small quantities of atmospheric nitrogen on artificial
media. 80 As much as 1.2 mgm. nitrogen may be fixed per 100 cc. of
medium. 81 Bac. asterosporus can fix 1 to 3 mgm. nitrogen for every

. 77 Pringsheim, H. fiber die Verwendung von Cellulose als Energiequelle zur
Assimilation des Luftstickstoffs. Centrbl. Bakt. II, 23: 300 308. 1909; 26:
222-227. 1910; Biol. Centrbl. 31: 65. 1911; Hutchinson and Clayton, 1918
(p. 196).

78 Stapp, C, and Ruschmann, G. Zur Biologie von Azotobacter. Arb. Biol.
Reichsaust. Land. u. Forstw. 13: 305-368. 1924.

79 Lohnis, F. Beitrage zur Kenntnis der Stickstoffbakterien. Centrbl.
Bakt. II, 14: 582-604. 1905. Lohnis, F., and Pillai, N. K. fiber stickstoff-
fixierende Bakterien. II. Centrbl. Bakt. II, 19: 87-96. 1907; 20: 781-799.
1908.

80 Lohnis, 1910 (p. 28); Lipman and Fowler, 1915 (p. 129).

81 Fred, E. B. The fixation of nitrogen by means of Bacillus radicicola without
the presence of a legume. Va. Agr. Exp. Sta. Ann. Rpt. 1909-1910, 138-142.

BACTERIA FIXING ATMOSPHERIC NITROGEN 121

gram of glucose consumed; the organism soon loses this property, but
it can regain it on passage through soil. 82 Planobacillus nitrofigens,
a large, rod-shaped, spore-forming bacterium, capable of fixing in three
weeks in a soil extract medium containing 2 per cent of mannite, 3.57
nigra, of nitrogen in 100 cc. of solution, was described. 83 A form
related to B. vulgar -e was found capable of fixing 1.8 to 4.7 mgm. of
nitrogen for one gram of sugar consumed. 84 A nitrogen-fixing organism
(Bac. azophile) was also isolated from manure. 85 Certain thermophilic
bacteria, growing in mixed culture at 61°C, are capable of fixing
appreciable quantities of nitrogen. 86

Azotobacter may also live symbiotically with algae 87 and other
bacteria. 88 The quantities of nitrogen thus fixed may be considerable. 89
The symbiotic action between CI. pastorianum and Azotobacter, where-
by the second uses up the oxygen making conditions favorable for the
former, has been demonstrated. 90 The various acids produced by the
former are neutralized by the soil bases and can be utilized by the
Azotobacter as sources of energy; this symbiotic action leads to a
maximum economy in the utilization of energy.

In respect to non-symbiotic nitrogen-fixation in the soil, Wino-
gradsky 91 distinguishes three catagories: 1. Very active soils are charac-
terized by an abundance of aerobic nitrogen-fixing bacteria and by the
ability to give readily spontaneous cultures of these bacteria when
enriched with an assimilable carbon source. 2. Soils only moderately

62 Bredemann, G. Untersuchungen ilber die Variation und das Stickstoff-
bindungsvermogen des Bacillus asterosporus A. M. ausgefiihrt an 27 Stammen
verschiedener Herkunft. Centrbl. Bakt. II, 22: 44-89. 1908.

83 Bondorff, K. A. Planobacillus nitrofigens n. sp. Den. Kgl. Veterinaer og
Landboholskr. Aarskr. 1918, 365-369.

84 Truffaut, G., and Bezssonoff, N. Un nouveau bacille fixant d'azote. Compt.
Rend. Acad. Sci.175: 544. 1922.

85 Fulmer, H. L., and Fred, E. B. Nitrogen-assimilating organisms in manure.
Jour. Bact. 3: 422-434. 1917.

86 Pringsheim, H. Ueber die Assimilation des Luftstickstoffs durch thermo-
phile Bakterien. Centrbl. Bakt. II, 31: 23-27. 1911.

87 Fischer, 1904 (p. 105); Heinze, B. Einige Beitrage zur mikrobiologischen
Bodenkunde. Centrbl. Bakt. II, 16: 640-653, 703-711. 1906.

88 Beijerinck and van Delden, 1902 (p. 105).

89 Hutchinson, C. M. Soil biology. Agr. Res. Inst. Pusa., Sci. Rpts. 1922-23,
43^9.

90 Omeliansky, 1916 (p. 109).

91 Winogradsky, S. Etudes sur Ies microbes fixateurs d'azote. Ann. Inst.
Past. 40: 455-520. 1926.

122 PRINCIPLES OF SOIL MICROBIOLOGY

active show less development of nitrogen-fixing bacteria and a re-
tarded or no development of spontaneous cultures. 3. Inactive soils
are free from aerobic nitrogen fixing bacteria. The methods of analyses
are described elsewhere (p. 732).

Symbiotic nitrogen-fixation by nodule bacteria. Historical. Many
centuries before the discovery of the nodule bacteria and their part in
the enrichment of the soil with combined nitrogen, due to their symbiotic
action with leguminous plants, had been established, the practical
agriculturist came to consider the growth of these plants in the soil
equivalent to manuring the soil for the succeeding crop. 92

In the early part of the 19th century Davy 93 stated that "peas and

beans seem to be well adapted to prepare the soil for wheat

Peas and beans contain a substance similar to proteins; but it seems that
the nitrogen, which is one of the constituents of this substance, takes its

92 Use of legumes by Romans is found in the work of Plinius-Historia naturalis
LVIII; Varro-De re rustica. Lib. I, Chap. 23; by ancient Chinese, in the book
of F. H. King — Farmers of forty centuries. Madison, Wis. 1911.

93 Davy, H. Elements of agricultural chemistry. 1814, p. 412.

PLATE VII
Symbiotic Nitrogen Fixing Bacteria

37. Types of nodules on leguminous and non-leguminous plants: A, Trifolium
pratense; B, Garden pea; C, Cowpea; D, Soja hispida; E, Vicia faba; F, Ceano-
thus americanus; G, nodules on leaf of Paivetta indica; H , Alnus. (A, after Mak-
rinov and Omeliansky; C, D, after Albrecht; E, after Straszburger and Omelian-
sky; F, after Burrill; G, after Faber and Omeliansky; II, after de Rossi.)

38. Detailed examination of Bad. radicicola on lupines : a, nodule, natural size;
b, cross section of root and nodule; c, cell of nodule filled with bacteria, X 400;
d, nodule bacteria, X 1200; e-/, bacteroids, X 1200 (after Woronin, Fischer and
Omeliansky).

39. Nodule bacteria of Vicia saliva: a-d, transformation of rods into bacteroids
(after Beijerinck).

40. Nodule bacteria from nodules of alfalfa, X 1200 (from Edwards and
Barlow).

41. Flagellation of bacteria of leguminosae: 1, Phaseolus vulgaris; 2, Cracca
virginiana; 3, Vicia sativa; 4, Medicago saliva; 5, Melilolus alba; 6, Lespedeza
striata (from Shunk).

42. Bad. rubiacearum of Pavetta zimmermanni: A, contents of nodule; B,
preparation of colony grown on agar; C, various forms of different stages of devel-
opment of pure culture: A and B, X 1660; C, X 1300 (after von Faber and de
Rossi).

PLATE VII

2. >»

ff -4

>/

-

B C

42

BACTERIA FIXING ATMOSPHERIC NITROGEN 123

origin from the atmosphere." Boussingault 94 was the first to carry out
a series of systematic studies on the nitrogen nutrition of leguminous
and cereal plants. He established the fact that, in the cultivation of
clover in unmanured soils, there is a definite gain, not only of carbon,
hydrogen and oxygen, but also of large quantities of nitrogen; wheat,
however, under the same conditions shows no gain or loss in nitrogen.
Boussingault definitely expressed his opinion that nitrogen belongs to
those elements which leguminous plants (clover, peas) can assimilate from
the atmosphere, while cereal plants (wheat, oats) cannot do so. In
attempting to repeat these experiments under more carefully controlled
conditions, Boussingault ignited the sand (thus killing the bacteria) and
found that neither cereals nor legumes were capable of assimilating
atmospheric nitrogen. 95

In an elaborate series of experiments begun in 1857 at the Rothamsted
Experimental Station, Lawes, Gilbert and Pugh 96 were so careful
to eliminate any possibility of the plants obtaining any combined
nitrogen from the atmosphere, that they destroyed the organism fixing
the nitrogen symbiotically with leguminous plants; they thus failed to
become the discoverers of this symbiotic relationship, since, in the
absence of the bacteria, the leguminous plants behaved like the cereals
and could not utilize the atmospheric nitrogen. Breitschneider 97
demonstrated in 1861 that legumes do not fix any nitrogen when the soil
is ignited but do so in unignited soil. Schulz-Lupitz 98 grew lupines for
fifteen consecutive times, without the application of nitrogen fertilizer
and without diminishing yields; cereals following lupines gave much
higher yields than on the same land not preceded by the leguminous
crop; the nitrogen content of the soil was actually found to increase.

The presence of nodules on the roots of leguminous plants was re-
corded by Malpighi 99 as early as 1687, but he, as well as others, con-

94 Boussingault. Recherches chimiques sur la vegetation enterprises dans le
but d'examiner si les plantes prennent de l'azote de l'atmosphere. Compt. Rend.
Acad. Sci.6: 102-112. 1838; 7: 889-892; Ann. Chim. et phys. (2), 67: 1-54 1838;
69: 353-367. 1838; Compt. Rend. Acad. Sci. 38: 580-607. 1854; 39: 601-613.

96 Ville. Note sur l'assimilation de l'azote de l'air par les plantes. Compt.
Rend. Acad. Sci. 31: 578. 1850; 35: 464-468, 650-654. 1852; 38: 705-709, 723-
727. 1854;43:143-148. 1856.

96 Lawes, Gilbert and Pugh, 1861 (p. 106).

97 Breitschneider. Kann der freie Stickstoff zur Bildung der Leguminosen
beitragen? Jahresber. Agr. Chem. 4: 123. 1861.

98 Schultz, L. Reinertrilge auf leichtem Boden, ein Wort der Erfahrung, zur
Abwehr der wirtschaftlichen Noth. Landw. Jahrb. 10: 777-848. 1881.

99 Malpighi. Opera omnia. Anatomia plantarum, Pars II. De gallis. 1687,
126.

124 PRINCIPLES OF SOIL MICROBIOLOGY

sidered them as root galls. Lachmann 100 observed, in 1858, that motile
bacteria cause the formation of the nodules and he suggested that the
nodules are the organs of nitrogen fixation. In 1866 Woronin 101 found
the nodules to consist of bacteria, but even he considered these nodules
as pathological outgrowths. Frank 102 demonstrated in 1879 that the
formation of nodules can be prevented by the sterilization of the soil.
Frank's view as well as that of other investigators 103 was that the
nodules are caused by outside infection. Hellriegel and Wilfarth 104 and
Atwater 105 • 106 finally demonstrated in 1884-1886 that the nodules on
the roots of leguminous plants are due to bacterial infection, that this is
beneficial, since it is within these nodules where the bacteria fix the
atmospheric nitrogen. When nodules were formed, the plants could
be grown on artificial soils containing but traces of combined nitrogen,
provided the mineral elements necessary for the nutrition of the
plant were present. In the absence of nodules, the plants were unable
to utilize the atmospheric nitrogen for its growth. When sterilized
soil was treated with fresh soil infusion, nodule formation took place
and the plants grew normally. The growth of the Gramineae de-
pended, however, on the nitrate content of the soil. These results
were soon confirmed by Lawes and Gilbert 107 and others.

100 Lachmann. tlber Knollchen der Leguminosen. Landw. Mitt. Zeitschr*
K. Lehranstalt. u. Vers. Sta. 1858, p. 37.

101 Woronin, M. Observations sur certaines excroissances que presentent les
racines de l'aune et du lupin des jardins. Ann. Sci. Nat. Bot., ser. 5, 7: 73-86.
1867; also Mem. Acad. Imp. Sci. St. Petersberg, 7 ser., 10: 1-13. 1866.

102 Frank, B. Uber die Parasiten in der Wurzelanschwellung der Papiliona-
ceen. Bot. Ztg. 37: 377-388, 393-400. 1879.

103 Ward, M. On the tubercular swellings on the roots of Vicia faba. Phil.
Trans. Roy. Soc. London 178, 1887.

104 Hellriegel, H. Welche Stickstoffquellen stehen der Pflanze zu Gebote?
Tagebl. Natforsch. Vers. Berlin, 1886, p. 290; Chem. Centrbl. 1886, 871; Landw.
Vers. Sta. 33: 464-465. 1886. Hellriegel, H., and Wilfarth, H. Untersuchungen
uber die Stickstoff-Nahrung der Gramineen und Leguminosen. Beilageheft
Ztschr. Ver. Riibenzuckerind. 1888, 1-234.

105 Atwater, W. O. On the assimilation of atmospheric nitrogen by plants.
Rpt. Brit. Assn. Adv. Sci. 54: 685. 1884.

106 Atwater, W. O., and Woods, C. D. The acquisition of atmospheric nitrogen
by plants. Amer. Chem. Jour. 6: 365. 1885; also 8: 398-420. 1886; Conn.
(Storrs) Agr. Exp. Sta. Bui. 5, 1889; Conn. (Storrs) Agr. Exp. Sta. Ann. Rpt. 1889.
11-51.

107 Lawes, J., and Gilbert, J. New experiments on the question of fixation of
free nitrogen. Proc. Roy. Soc. London 47: 85-118. 1890.

BACTERIA FIXING ATMOSPHERIC NITROGEN 125

The causative organism was isolated in 1888, in pure culture, by Bei-
jerinck, 108 who named it Bacillus radicicola. Beijerinck described three
stages in the development of the organism.

1 . The organism is present in the soil in the form of small rods which
can penetrate the root hairs of the leguminous plants and from there it
is transferred to the "infectious tissue."

2. The organism changes into a motile bacillus.

3. It changes into the bacteroid form which functions as the sym-
biotic organism.

The organism was soon grown, on artificial culture media, by a
number of investigators. 109 The mechanism of root infection by pure cul-
tures of bacteria was worked out by Prazmowski in 1889. 109a Schloesing
and Laurent 110 demonstrated that the nitrogen is actually obtained by
the bacteria in the form of nitrogen gas from the atmosphere. Legumi-
nous plants were grown in sterile glass cylinders containing sterile
sand and watered with sterile water. When the composition of the gas
in the cylinder was determined, it was found that, while the uninoculated
plants showed a gain of only 0.6 mgm. of nitrogen and no nodule forma-
tion, inoculated plants showed a gain of 34.1 and 40.6 mgm. of nitrogen
and abundant nodule formation. Nobbe and Hiltner 111 concluded that
the fixation of nitrogen by leguminous plants is closely related to the
formation of bacteroids in the nodules.

Nomenclalure. The causative organism of the nodules on the roots of
leguminous plants is referred to by different names, depending on the
particular system of classification. As pointed out above, the discoverer

108 Beijerinck, 1888 (p. 103). See also Prazmowski. Das Wesen und die
biologische Bedeutung der Wurzelknollchen der Erbse. Bot. Centrbl. 39: 356-
362. 1889; Landw. Vers. Sta. 37: 161-238. 1890.

109 For a review of earlier literature see Voorhees and Lipman, 1907 (p. 491).
Hiltner, 1904 (p. 128). Lohnis, 1910. Burrill and Hansen, 1917 (p. 126); Miiller,
A., and Stapp, C. Beitriige zur Biologie der Leguminosenknollchenbakterien
mit besonderer Beriicksichtigung ihrer Artverschiedenheit. Arb. Biol. Reich-
sanst. L. u. Forstm. 14: 455-554. 1925.

loss Prazmowski, A. Die Wurzelknollchen der Erbse. Landw. Vera. Sta. 37:
161; 38: 5. 1890.

110 Schloesing, Th., and Laurent, E. Recherches sur la fixation de l'azote
libre par les plantes. Compt. Rend. Acad. Sci. Ill : 750-754. 1890; 113 : 776-778,
1095-1060. 1891; 115: 1017. 1892; Ann. Inst. Past. 6: 65-115, 824-940. 1892.

111 Nobbe, F., and Hiltner, L. Wodurch werden die knollchenbesitzenden
Leguminosen befahigt, den freien atmospharischen Stickstoff fur sich zu ver-
werten. Landw. Vers. Sta. 42: 459-478. 1893.

126 PRINCIPLES OF SOIL MICROBIOLOGY

of the organism, Beijerinck, termed it Bacillus radicicola. In view of the
fact that this is a non-spore forming organism and it is destroyed at
60° to 70°C, Prazmowski changed its name to Bacterium radicicola.
The fact that a number of races produce only a single polar fiagellum
led various investigators 112 ,u3 to classify the organism with the genus
Pseudomonas, under the name of Pseudomonas radicicola. E. F. Smith 114
and the Committee of the Society of American Bacteriologists (p. 58)
decided that the organism described by Frank 115 in 1879 as Schinzia
leguminosarum was the nodule forming organism and deserves priority;
the name of Bacterium leguminosarum or Rhizobium leguminosarum
was therefore suggested. It is doubtful, however, whether Frank
ever saw the nitrogen-fixing, nodule-forming organism. 116 According
to Lohnis and Hansen, 117 the nodule bacteria do not represent a special
genus Rhizobium, but are closely related to Bad. radiobacter, Bact.
lactis viscosum, Bact. pneumoniae and Bact. aerogenes, the last three
being immotile and the first motile. The species differ only to a slight
extent, in their physiological and morphological characters; the
branched cell forms (so-called "bacteroids") are common to all mem-
bers of the group of capsule bacteria, when tested adequately. These
closely related forms are well distributed in the soil and Bact. radio-
bacter may actually be present in the root nodules of leguminous plants.
On account of its resemblance to Bact. radicicola, it has been mistaken
for the nodule-producing organism in the cowpea-soybean group, since
it grows rapidly on the plates made from the nodules; however, it can be
differentiated from the latter by its brown growth on potato.

Media. A number of media have been suggested, at various times,
for the cultivation of the organism causing the nodules on leguminous

112 Moore, G. T. Bacteria and the nitrogen problem. Yearb. U. S. Dept. Agr.
for 1902, 333-342.

113 Burrill, T. J., and Hansen, R. Is symbiosis possible between legume bac-
teria and non-legume plants? 111. Agr. Exp. Sta. Bui. 202: 115-181. 1917.
(Complete bibliography to 1915).

114 Smith, E. F. Bacteria in relation to plant diseases. Washington, 2: 97-
146. 1921.

118 Frank, B. Uber den gegenwartigen Stand unserer Kenntnis der Assimila-
tion elementaren Stickstoffs durch die Pflanze. Ber. deut. bot. Gesell. 7: 234-
247. 1889; Landw. Jahrb. 19: 523-640. 1890; also Frank, 1879 (p. 124).

116 Kellerman, K. F. The present status of soil inoculation. Centrbl. Bakt.
II, 34: 42-50. 1912.

117 Lohnis, F., and Hansen, R. Nodule bacteria of leguminous plants. Jour.
Agr. Res. 20: 543-556. 1921.

BACTERIA FIXING ATMOSPHERIC NITROGEN 127

plants. In addition to various organic media, extracts of carrots, of
leaves and of seeds of leguminous plants, a number of inorganic
media have been suggested. Of these, several may be selected:

1. Wood ash medium: 118

Wood ash extract (15 grams ashes to 1 liter of tap water) . . 1000 cc.

Sucrose 10 grams

KH 2 P0 4 3 grams

2. Ashby's mannite solution (p. 113J.

3. Conn's asparaginate solution (p. 16)

4. Glucose medium: 119

Distilled water 1000 cc. NaCl Trace

Glucose 20 grams FeS0 4 Trace

KH 2 P0 4 l.Ogram MnS0 4 Trace

MgS0 4 -7H 2 O.lgram CaCl 2 Trace

5. Sucrose medium:

Tap water 1000 cc. KH 2 P0 4 1.0 gram

Sucrose 10 grams MgS0 4 0.5 gram

6. Mannite medium: 120

Mannite 10 grams CaC0 3 l.Ogram

NaCl 0.2 gram Yeast water 100 cc.

K 2 HP0 4 0.5 gram Distilled water 900 cc.

MgSO 4 .7H 2 0.2 gram Washed agar 15 grams

CaS0 4 -2H 2 0.1 gram

The yeast water is prepared 121 by stirring starch-free yeast with ten times its
weight of tap water, steaming for 1 to 2 hours, then sterilizing and, after allowing
to stand 24 hours, siphoning off the clear brown liquid.

Various legume extract and tomato extract media are also employed: A decoc-
tion of 100 grams material of the green plants and roots in 1000 cc. of water, to
which 1 per cent glucose is added and some CaC0 3 to make the reaction neutral. 1 ' 22
For solid media, 1.2 to 1.5 per cent of agar is used; for gelatin media, 12 per
cent of gelatin is used. When the reaction is adjusted by the hydrogen-ion con-
centration method, it should be brought to pH 6.8 to 7.5.

118 Harrison, F. C, and Barlow, B. The nodule organism of the Leguminosae—
its isolation, cultivation and commercial application. Centrbl. Bakt. II, 19:
264-272, 426^41. 1907; Trans. Roy. Soc. Can. Ser. (2), 12: 157-237. 1907.

119 Fred, E. B. A physiological study of the legume bacteria. Va. Agr. Exp.
Sta. Ann. Rpt. 1911, 145-174.

120 Wright, W. H. The nodule bacteria of soybeans. I. Bacteriology of
strains. Soil Sci. 20: 95-120. 1925.

121 Fred, E. B., Peterson, W. H., and Davenport, A. Fermentation character-
istics of certain pentose destroying bacteria. Jour. Biol. Chem. 42: 175. 1920.

122 Nobbe, F., and Hiltner, L. Kunstliche Ueberfuhrung der Knollchenbak-
terien von Erbsen in solche von Bohnen (Phaseolus). Centrbl. Bakt. II, 6: 449-
457. 1900.

128 PRINCIPLES OF SOIL MICROBIOLOGY

Nodule formation. The bacteria usually enter the plant through the
root hairs, being attracted through the secretion of soluble carbohydrates
or organic acids by the plant. On entering the root, the bacteria
multiply forming a thread of infection, similar to a fungus hypha, which
enters the root and branches out into the parenchymatous cells of the
plant. In some cells, the thread breaks up into individual cells
which, on multiplication, fill the whole protoplasm of the cell; the
bacteria give rise at the same time to branching forms, commonly
referred to as "bacteroids."

The size, form and position of nodules vary with the nature of the
plant, soil in which it is grown and virulence of the bacteria, as shown by
Hiltner, 123 who explained nodule formation by his theory of immunity
discussed elsewhere (p. 589). According to Bryan, ui nodule formation
is greatly influenced by the reaction of the soil : alfalfa and clover produce
maximum growth and number of nodules at pH 7.8, alsike and red
clover at pH 5.6; the critical pH values for nodule formation are 4.0 and
9.0 to 10.0. Nodules will be formed at all temperatures at which the
plant can make a growth that is at all vigorous. 125 The presence of
nitrates or other available nitrogen compounds in the soil depresses
nodule formation.

Isolation of organism from nodules. Harrison and Barlow 126 describe
in detail the method of isolation and cultivation of the organism.

A medium sized nodule, appearing young and sound, is selected. It is cut off
so as to leave 2 to 3 mm. of the root on both sides to permit handling it with forceps.
The nodule is then washed, rinsed in distilled water and dropped into a sterilizing
liquid containing 1 gram HgCl 2 and 2.5 cc. c. p. HC1 in 500 cc. of water. The
nodule is well shaken in the solution for 3 to 4 minutes, then washed three times
in sterile distilled water. It is then covered with about 1 cc. of sterile distilled
water and crushed with a sterile, heavy glass rod. Two or three drops of the
cloudy suspension are placed into a test tube of the agar medium, which has
previously been liquefied and cooled to 45°C. A second tube of agar is then in-
oculated with five drops from the first; a third tube is inoculated from the

123 Hiltner, L. Die Bindung von freiem Stickstoff durch das Zusammenwirken
von Schizomyceten und von Eumyceten mit hoheren Pflanzen. Lafar's Handb.
techn. Mykol. 3: 24-70. 1904.

124 Bryan, O. C. Effect of reaction on growth, nodule formation and calcium
content of alfalfa, alsike clover and red clover. Soil Sci. 15: 23-35. 1923.

126 Jones, F. R., and Tisdale, W. B. Effect of soil temperature upon the de-
velopment of nodules on the roots of certain legumes. Jour. Agr. Res. 22:
17-31. 1921.

1,6 Harrison and Barlow, 1907 (p. 127).

BACTERIA FIXING ATMOSPHERIC NITROGEN 129

second and a fourth tube from the third; the plates are poured and incubated
at 20° to 25°C. The organism is isolated upon sterile agar slants or liquid
media from a typical colony upon the plate, using the third and fourth plates
and discarding the first two. The lens-shaped and pin-head colonies should be
selected rather than the giant colonies. In case of questionable plates, replating
may be necessary from the culture isolated. To keep the cultures in stock,
one of the above agar media (ash or mannite agar) may be used.

Isolation from soil. The Bad. radicicola can readily spread through
the soil 127 and persist there for a long period of time. The bacteria
move in the soil at a definite rate. 128 Bad. radicicola can also be cul-
tivated from the soil, although the specificity of the forms isolated by
Nobbe and Hiltner and Gage 129 has not been sufficiently demon-
strated. The results of Greig-Smith concerning the great abundance of
Bad. radicicola in the soil were not confirmed. The numbers of each
strain in the soil depend upon the reaction of the soil, an acidity
greater than pH 5.4 being detrimental to the development of most
strains; at a favorable reaction (pH 5.4-6.8, depending on strain), as
many as 100,000 to 1,000,000 cells of different strains may be found
per gram of soil. 130 Kellermann and Leonard 131 could isolate the
organism only from soils sterilized and previously inoculated. Lip-
man and Fowler 132 isolated Bad. radicicola from soil, in which le-
gumes have previously been grown, and demonstrated its ability to
cause the formation of nodules on the roots of leguminous plants.
Two media were employed: (1) 1000 grams of water, 10 grams
maltose, 1 gram K 2 HP0 4 , 1 gram MgS0 4 , 2 to 3 drops each of 10 per
cent solution of NaCl, FeCl 3 , MnS0 4 , and CaCl 2 and 15 grams of
agar. (2) Soil extract, obtained by boiling one part of soil with
three parts of water for one hour, then filtering and adding 15 grams of
agar and 10 grams of maltose to 1 liter of the extract. A soil in which

127 Ball, O. M. A contribution to the life history of Bacillus (Ps.) radicicola
Beij. Centrbl. Bakt. II, 23: 47-59. 1909.

128 Kellerman, K. F., and Fawcett, E. H. Movements of certain bacteria in
soils. Science, 25: 806. 1907.

129 Nobbe, Hiltner and Schmid, 1895 (p. 134); Gage, G. E. Biological and
chemical studies on nitrosobacteria. Centrbl. Bakt. II, 27: 7-48. 1910.

130 wn S on, J- K. Legume bacteria population of the soil. Jour. Amer. Soc.
Agron. 18:911-919. 1926.

131 Kellerman, K. F., and Leonard, L. T. The prevalence of Bacillus radici-
cola in soil. Science, n. s. 38:95-98. 1913.

132 Lipman, C. B., and Fowler, L. W. Isolation of Bacillus radicola from
soil. Science, N. S. 41: 256-259, 725. 1915.

130 PEINCIPLES OF SOIL MICROBIOLOGY

Vicia sicula has been grown a year before was used for plating out on
these media. The capacity of the colonies developing on the plate to
inoculate plants obtained from disinfected seed grown in sterile soil was
then tested, and it was found that nearly half of the colonies were those
of the organism in question.

Vogel and Zipfel 133 demonstrated by agglutination tests, using highly
potent immune serum, that the nodule bacteria can be readily isolated
from the soil; this method is even more reliable than the direct inocula-
tion test, since, with the latter method, negative inoculation results are
often obtained.

Colony appearance. The colonies appearing on the plate are either
surface or deep colonies. The first are drop-like, watery, mucilaginous
in appearance, gray-white to pearly white in color, glistening, and semi-
translucent to opaque. The edges are smooth and even ; they frequently
attain a size of 1 cm. or more in diameter. The deep colonies are small,
lens or spindle shaped, with smooth and even edges, opaque, granular in
structure, and cream colored to chalky white. They slowly increase in
size, eventually appearing on the surface, when growth becomes rapid.
When first isolated, colonies may not appear before 6 to 14 days. Some
races grow much faster than others, as in the case of Pisum, Vicia,
Lupinus, Trifolium, Melilotus, and Medicago. To the slow growers
belong the Vigna (cowpea), Glycine (Soja, soybean), and others (No.
46, PI. VIII).

Morphology and life cycle of organism. The organism varies greatly
in size and shape in the nodule. Many small, oval forms, described by
Beijerinck as swarmers, and normal rods are found together with a few
large club-shaped or branching forms (bacteroids) in the young nodules.
In the old, decomposing nodule, the branching forms are extremely
vacuolated, showing small, oval, deep staining bodies within. 134 These
bodies may be the motile swarmers or the branching form dividing into
bacilli.

In pure cultures, the organism forms minute short rods, motile when
young by means of flagella. 135 The bacteroids may be produced also

133 Vogel, J., and Zipfel, H. Beitrage zur Frage der Verwandtschaftsverhalt-
nisse der Leguminosenknollchenbakterien und deren Artbestimmung mittels
serologischen Untersuchungsmethoden. Centrbl. Bakt. II, 54: 13-34. 1921.

134 de Rossi, G. Uber die Mikroorganismen welche die Wurzelknollchen der
Leguminosen erzeugen. Centrbl. Bakt. II, 18: 289-314, 481^89. 1907.

136 Barthel, Chr. Die Giesseln des Bacterium radicicola (Beij.). Ztschr. Gar-
ungsphys. 6: 13. 1917.

BACTERIA FIXING ATMOSPHERIC NITROGEN 131

on artificial culture media in the presence of acid phosphate, 136 sodium
succinate and glycerol, 137 caffeine 138 and cumarine. 139 According to
Barthel, 140 caffeine and other vegetable alkaloids, like guanidine, pyridine
and chinoline, will stimulate the formation of involution forms in pure
culture; he suggested, therefore, that the formation of these so-called
bacteroids in root nodules is due to the presence of alkaloids in the plant.
The bacteroids are never so large and numerous on the artificial culture
media as in a young nodule; they are produced, either in the medium,
or in the nodule due to specific nutrition or to unfavorable conditions;
in that stage they are hardy and multiply rapidly. According to Zipfel,
the branching forms are not degeneration forms, but may be looked
upon as a normal and necessary stage in the life of the organism with
specific biological functions; they are formed from rods and change
again into rods when inoculated into proper media.

Five stages in the life cycle of the Bad. radicicola, through which it
passes under cultural conditions, were recognized. 141

1 . Non motile, pre-swarmer form, obtained in 4 to 5 days when a culture of the
organism is placed in a neutral soil solution.

2. Larger, non-motile coccus. The pre-swarmer coccoid changes in the pres-
ence of saccharose, certain other carbohydrates and phosphates, by increasing in
size until the diameter has doubled.

3. Motile, swarmer stage, when the cell becomes ellipsoidal and develops high
motility.

4. Rod-form, as a result of the further elongation of the swarmer, with decreas-
ing motility.

5. Vacuolated stage. When available carbohydrates become exhausted or the
organism is placed in a neutral soil extract, the cell becomes highly vacuolated and
the chromatin divides into a number of bands. Finally these bands become
rounded off and escape from the rod as the coccoid pre-swarmer. The pre-
swarmer stage is usually formed from normal rods in calcareous soils, when

136 Stutzer, A. Die Bildung von Bakteroiden in kiinstlichen Nahrboden.
Centrbl. Bakt. II, 7: 897-912. 1901.

137 Buchanan, R. E. The bacteroids of Bacillus radicicola. Centrbl. Bakt. II,
23: 59-91. 1909.

138 Zipfel, H. Beitrage zur Morphologie und Biologie der Knollchenbakterien
der Leguminosen. Centrbl. Bakt. II, 32: 97-137. 1912.

139 Fred, 1911 (p. 127).

140 Barthel, C. Contribution a la recherche des causes de la formation des
bacteroides chez les bacteries des Legumineuses. Ann. Inst. Past. 35: 634-647.
1921.

141 Bewley, W. F., and Hutchinson, H. B. On the changes throughwJufllLthe
nodule organism (Ps. radicicola) passes under cultural conditionfl^JtyWr./&gr>\
Sci. 10: 144-162. 1920. /<^O or ' *o VN

» -ft &\

ujl LIBRARY 3QJ

132 PRINCIPLES OF SOIL MICROBIOLOGY

calcium or magnesium carbonates are added to the medium, or under anaerobic
conditions. Acid soils cause the production of highly vacuolated cells and
eventually kill the organism. These studies need further confirmation.

Motility. In young agar slants, the organisms are found to be very
motile. Owing to the slime produced by the organism, the demonstra-
tion of flagella is very difficult; this was the reason for considerable
disagreement among the different investigators. It has come to be
recognized, 142 however, that the nodule bacteria possess two types of
flagellation: peritrichous and monotrichous. Differences, however,
have been reported even for a single strain. The soybean organism
was reported 143 by some as possessing peritrichic flagellation, but by
most other workers 144 as monotrichous. The differences thus obtained
were due either to the fact that cultures of various ages were employed
or different types of bacteria exist, even for the same plant (as Soja
max), in different parts of the world. 145 ' 146

For staining of flagella, the following modification of the Loeffler's stain may be
used :

Solution A

parts

Ferric chloride (1 : 20 aqueous solution) 1

Saturated aqueous solution of tannic acid 3

This solution improves with age; it should be at least a week or two old and

should be filtered before using.

Solution B

parts

Anilin oil 1

95 per cent alcohol 4

The bacterial suspension is allowed to air-dry on a clean cover glass. About
5 drops of solution A are then placed on the cover glass, followed immediately
by 1 to 2 drops of solution B. The combination is allowed to act at room tem-
perature for 2 minutes and is then washed in distilled water. The stain (30 parts
of saturated alcoholic solution of methylene blue, 13 parts of solution B as mord-
ant and 100 parts of 1 : 10,000 KOH solution) is applied for 2 minutes.

142 Hansen, R. Note on the flagellation of the nodule organisms of the Legum-
inosae. Science. N. S. 50: 568-569. 1919.

143 Wilson, J. K. Physiological studies of Bacillus radicicola of soybean {Soja
max Piper) and of factors influencing nodule production. Cornell Univ. Exp.
Sta. Bui. 386. 1917.

144 Wright, 1925 (p. 127).

145 Shunk, I. V. Notes on the flagellation of nodule bacteria of leguminosae.
Jour. Bact. 6: 239-246. 1921; Ibid. 5: 181-187. 1920.

140 Fred and Davenport, 1918 (p. 582).

BACTERIA FIXING ATMOSPHERIC NITROGEN 133

Lohnis and Hansen and Shunk observed the two distinct types of
flagellation referred to above. In the single flagellate types (monotri-
chous) , the flagellum is not strictly polar but is usually attached to the
corner. However, organisms obtained from nodules of different species
of plants belonging to one genus have the same type of flagellation.

Physiology of nodule bacteria. The different strains of Bad . radicicola
are strictly aerobic.

Maltose, sucrose, glucose and mannite offer the best sources of
carbon; lactose, dextrin and glycerol can also be utilized. According
to Beijerinck, separate carbon and nitrogen sources (asparagine,
ammonium sulfate, sodium or potassium nitrate) are required.
Laurent 147 first showed that the organism can be cultivated on nitrogen-
free media, containing 0.1 per cent KH 2 P0 4 , 0.01 per cent MgS0 4 and
5 to 10 per cent of an available energy source. When grown on such
a medium, it will fix atmospheric nitrogen. 148

The presence of nitrates in the medium and in the soil diminishes
nitrogen-fixation by the organism. This has been demonstrated by
Nobbe and Richter 149 and others, and it was found to be due not to any
injurious influence of the nitrate but to the fact that the plant, capable of
obtaining its nitrogen from the soil, represses the development of the
nodules.

A condition is found here very similar to the influence of nitrates
upon nitrogen fixation by non-symbiotic bacteria. Prucha 150 found
that the addition of KN0 3 , Ca(N0 3 ) 2 , NH 4 C1, or peptone to sandy
soil, at the rate of 0.25 gram of the salts to 300 grams air-dry soil, had an
inhibiting effect on nodule development of Canada field pea, while
MgS0 4 , KH 2 P0 4 , Ca (H 2 P0 4 ) 2 and tannic acid, especially in low
concentrations, had a beneficial effect.

The optimum reaction for the growth of the bacteria is pH 5.5 to 7.0,
depending on the nature of the plant, with limiting reactions of pH
3.2 to 5.0 on the acid side, and pH 9.0 to 10.0 on the alkaline. The
optimum temperature is 25° to 28°C. with 0° and 50° as the limits.

147 Laurent, E. Sur le microbe des nodosit6s des Lcgumineuses. Compt.
Rend. Acad. Sci. Ill: 754. 1890; Ann. Inst. Past. 4: 722. 1890; 5: 105-139.
1891.

148 Fred, 1910 (p. 120).

149 Nobbe, F., and Richter, L. Uber den Einfluss des Nitratstickstoffs und der
Humussubstanzen auf den Impfungserfolg bei Leguminosen. Landw. Vers. 56:
441-448. 1902; 59: 167-174. 1904.

150 p ruc ha, M. J. Physiological studies of Bacillus radicicola of Canada field
pea. Cornell Univ. Agr. Exp. Sta. Mem. 5, 1915.

134 PRINCIPLES OF SOIL MICROBIOLOGY

The nodule bacteria can be modified in their ability to grow under
unfavorable conditions; a character, such as tolerance to dyes, may
be modified relatively quickly (Burke and Burkey). 151 However,
the character which has been lost as a result of cultivation on artifi-
cial media is quickly regained when the culture is returned to the
soil.

Specific differentiation. Three groups of methods are usually em-
ployed for the specific differentiation of the nodule bacteria: (1) plant
inoculation, (2) morphological and cultural studies, (3) serological
and immunological reactions. Although Nobbe, Hiltner and Schmid 152
came to the conclusion that the bacteria in the nodules of all legumes
are strains of the same organism, the fact was soon brought to light
that not all the bacteria obtained from the nodules of various plants
can cross-inoculate and produce nodules on the roots of other leguminous
plants. These plants could readily be divided into several closely re-
lated groups, the plants belonging to each group having their own
specific organism, with cross inoculation taking place only by the mem-
bers of each group.

Hiltner and Stdrmer 153 came to recognize, on the basis of morphological
and cultural studies, two groups of nodule bacteria: (1) Bad. radicicola
on Pisum, Vicia, Lathyrus, Phaseolus, Trifolium, etc., and (2) Bact.
beijerinckii on Lupinus, Ornithopus, Glycine. The former grows well
on certain gelatin media and readily produces branching forms, while
the latter grows poorly on gelatin media. It was soon found that a
further subdivision would have to be made, Pisum, Trifolium, Medicago
and Lupinus bacteria being taken as representative types.

Zipfel 154,155 made use of agglutination tests and concluded that nodule
bacteria were not varieties of the same species, but that distinct species
existed. Six groups were thus distinguished: (1) Lupinus, (2) Trifolium,
(3) Medicago, (4) Pisum, (5) Faba, and (6) Phaseolus.

151 Burke, V., and Burkey, L. Modifying Rhizobium radicicolum. Soil Sci.
20: 143-146. 1925.

162 Nobbe, F., Hiltner, L., and Schmid, E. Versuche iiber die Biologie der
Knollchenbakterien der Leguminosen, insbesondere liber die Frage dei Arteinheit
derselben. Landw. Vers. Sta. 45: 1-27. 1895.

153 Hiltner, L., and Stormer, K. Neue Untersuchungen iiber die Wurzelknoll-
chen der Leguminosen und deren Erreger. Arb. k. Gesundhtsamt., Biol. Abt. 3:
151-307. 1903.

154 Zipfel, 1912 (p. 131).

165 Vogel and Zipfel, 1921 (p. 130).

BACTERIA FIXING ATMOSPHERIC NITROGEN 135

On the basis of serological investigation, Klimmer and Kruger 156
formed nine groups of legume bacteria: (1) Lupinus and Ornithopus,
(2) Melilotus, Medicago, and Trigonella, (3) Vicia (V. sativa), (4)
Pisum, (5) Vicia faba, (6) Trifolium pratense, (7) Phaseolus vulgaris,
(8) Soja hispida, and (9) Onobrychis sativa. 157 Other serological
studies 158 confirmed the general conclusion that the nodule bacteria
include more than one organism.

The agar test-tube method may be used for the study of nodule forma-
tion on the roots of legumes by different strains of bacteria. 159 On the
basis of the cultural method, the nodule bacteria were divided into the
following groups: (1) alfalfa organism inoculating also Medicago lupu-
lina, M. denticulata and Melilotus, (2) clover organism inoculating all
species of Trifolium, (3) vetch and garden pea, (4) cowpea, (5) soybean,
(6) garden bean. Burrill and Hansen 160 demonstrated, by cross-inocula-
tion studies, eleven kinds of bacteria divided into three groups, namely:
(1) thin, scant, slow growth on ash-agar slant; little gum formed,
flagella easily demonstrated — Vigna, Cassia, Acacia, Glycine, etc.; (2)
more rapid and more abundant growth; glistening, opaque and pearly
white; considerable gum formed which interferes with attempt of
staining flagella — Melilotus, Medicago, Trigonella; (3) very fast,
spreading growth; watery and semi-translucent; very slimy and sticky,
due to excess of gum — Vicia, Pisum, Lens, Lathyrus, Trifolium,
Phaseolus and Strophostyles.

Lohnis and Hansen 161 divided the bacteria of the leguminous plants
into two groups, the representatives of which differ both morphologi-
cally and physiologically. The first group shows all the features of
Bad. radicicola; it is peritrichic, grows relatively fast on agar plates and

156 Klimmer, M., and Kruger, R. Sind die bei den verschiedenen Legumino-
sen gefundenen Knollchenbakterien artverschieden? Centrbl. Bakt. II, 40:
256-265. 1914; Klimmer, M. Zur Artverschiedenheit der Leguminosen-Knoll-
chenbakterien festgestellt auf Grund serologischer Untersuchungen. Centrbl.
Bakt. II, 55: 281-283. 1922.

157 Simon, J. Uber die Verwandtschaftsverhaltnisse der Leguminosen-Wurzel-
bakterien. Centrbl. Bakt. II, 41: 470-479. 1914.

168 Stevens, J. W. Can all strains of a specific organism be recognized by agglu-
tination? Jour. Inf. Dis. 33: 557. 1923; A study of various strains of Bacillus
radicicola from nodules of alfalfa and sweet clover. Soil Sci. 20: 45-66. 1925.

169 Garman, H., and Didlake, M. Six different species of nodule bacteria.
Ky. Agr. Exp. Sta., Bui. 184: 343-363. 1914.

160 Burrill and Hansen, 1917 (p. 126).

161 Lohnis and Hansen, 1921 (p. 126).

136 PRINCIPLES OF SOIL MICROBIOLOGY

changes milk characteristically; it produces nodules on the roots of
clover, sweet clover, alfalfa, vetch, pea, navy bean, lupine, black locust,
Amorpha and Strophostyles. The second group is characterized by
monotrichic flagellation, comparatively slow growth on agar plates,
and inability to cause a marked change in milk. It has been isolated
from the soybean, cowpea, lima bean, peanut, beggarweed, Acacia,
Genista Cassia and Amphicarpa. However, they do not suggest sepa-
rating the organism into two new species before the complete life his-
tory of the two groups is known.

Bergey, 162 following the system proposed by the Society of American
Bacteriologists placed the Bad. radicicola in a separate genus
"Rhizobium," and separated the different forms into two species:

(1) Rh. leguminosum Frank, inoculating Pisum, Vicia, Lathyrus, etc.,

(2) Rh. radicicolum Beij. of Trifolium, Phaseolus, etc.

The following is a list of leguminous plants, divided on the basis of inter-
inoculation. 163 The different members in any one group are those which can be
inoculated by the strain of the Bact. radicicola specific for that group.

Group I: Group III:

Trifolium pratense, red clover Vigna sine7isis, cowpea

Trifolium hybridum, alsike clover Cassia chamaecrista, partridge pea

Trifolium alexandrinum, bersem Arachis hypogoea, peanut

clover Lespedeza striata, japan clover

Trifolium incarnalum, crimson clover Mucuna utilis, velvet bean

Trifolium repens, white clover Baptisia linctoria, wild indigo

Trifolium medium, zigzag, or cow Desmodium canescens, tick trefoil

clover Acacia armata, acacia

Group II: Genista tinctoria, dyer's greenwood

Melilotus alba, white sweet clover Phaseolus lunatus, lima bean

Melilotus officinalis, yellow sweet Group IV:

clover Pisum sativimi arvense, Canada field

Mcdicago sativa, alfalfa pea

Medicago hispida, bur clover Vicia villosa, hairy vetch

Medicago lupulina, black medick, or Vicia sativa, spring vetch

yellow trefoil Vicia faba, broad bean

Trigonella foenum-graccum, fenu- Lens esculenta, lentil

greek Lathyrus latifolius, sweet pea

162 Bergey, 1923 (p. x).

163 Hansen, R. Symbiotic nitrogen-fixation by leguminous plants with special
reference to the bacteria concerned. Scientific Agriculture (Canada) 1: 59-62.
1921; Whiting, A. L., Fred, E. B., and Helz, G. E. A study of the root nodule
bacteria of wood's clover (Dalea alopecuroides). Soil Sci. 22: 467-476. 1926.

BACTERIA FIXING ATMOSPHERIC NITROGEN 137

Group V: Group IX:

Glycine hispida (Soja max), soybean Amorpha canescens, lead plant

Group VI: Group X:

Phaseolus vulgaris, garden bean Strophostyles helvola, trailing wild

Phaseolus mullifiorus, scarlet runner bean

Group VII: Gr ° U P XI: „

Lupinus percnnis, lupine Robtma pseudo-acacia, black or com-

Ornithopus saliva, seradella mon locust

Group VIII: Group XII:

Amphicarpa monoica, hog peanut Dalea alopecuroidcs, wood's clover

An interchangeability between the soy bean and cowpea has been
demonstrated 164 in the laboratory, however. Field tests from other
sources do not indicate such relationship. Various explanations for the
specificity, based on soil reaction, climate, etc., have been proposed.
Burrill and Hansen properly suggested that it may be a case of specific
enzymes produced by the bacteria or of differences in the root-sap,
which cannot be detected by chemical methods. So far we have to
depend on cross inoculation and serological tests for the specific separa-
tion. No morphological differences have yet been established, ex-
cept the division into two groups suggested by Lohnis and Hansen;
we do not know whether we are dealing here with different species
or mere biological races.

The application of serological reactions has brought out the fact
that various strains of bacteria may form nodules on the same plant,
but only one serological type is found in the same nodule. 165 Other
investigators 166,167 also found that not all strains of Bad. radicicola
of one leguminous plant are identical. This suggested the existence of
various biotypes even for the same plant. The existence of two general
types of the organism which can form nodules on the soy bean, identical
morphologically but different physiologically and especially serologi-
cally, has been demonstrated. 167

These results are probably due to the fact that a bacterial culture is
actually a population in which the different cells have variable proper-
ties. Although morphology may not be sufficient to demonstrate any

164 Leonard, L. T. Nodule production kinship between the soybean and cow-
pea. SoilSci. 15: 277-283. 1923.

165 Bialosuknia, W., and Klott, C. Badania nad Baklerium radicicola. Roczn.
Nauk. Rolniczych. 9: 288-335. 1923.

166 Stevens, 1923-25 (p. 135).

167 Wright, 1925 (p. 127).

138 PRINCIPLES OF SOIL MICROBIOLOGY

differences between the members of the population, physiological reac-
tions and the even more sensitive serological reactions can bring out
these variations. This explains the modification of a strain when
grown on artificial culture media or as a result of repeated passage
through the host plant. It also suggests the possibility of improving
or deteriorating a strain by the proper selection of the types of cell.
This phenomenon explains the increase in activity and fixation of nitro-
gen by repeated passage through plants. 168 The process of adaptation
to a particular host plant is longer in case of vegetatively weak organ-
isms than for vegetatively strong organisms.

A detailed study of the chemistry of nitrogen fixation by nodule
bacteria (588) and the artificial inoculation of soil with bacterial cul-
tures (817) will be discussed elsewhere.

Nodule formation by non-leguminous plants. In addition to the
legumes, a number of non-legumes are found possessing nodules on their
roots. Of these, most attention has been paid to Ceanothus (red-root) ,
Elaeagnus (silver berry), Alnus (alder), Podocarpus, Cycas and Myrica
(sweet gale).

At first these nodules were thought to be of fungus origin. The
nodules of Alnus, Elaeagnus and Ceanothus were found 169-171 to be
caused by bacteria belonging to the Bad. radicicola group and capable
of fixing nitrogen. In some plants at least (Myrica) the organism is of
the nature of an Actinomyces. 172 Coriaria japonica produces nodules
similar to those produced by the Alder, due probably to an Actinomyces
(Act. myricae according to Peklo) in both cases. 173 In the roots of
cycads, Bad. radicicola, Azotobacter and an alga ( Anabaena) were demon-

168 Wunschik, H. Erhohung der Wirksamkeit der Knollchenerreger unserer
Schmetterlingsblutler durch Passieren der Wirtpflanze. Centrbl. Bakt. II, 64:
395-445. 1925.

169 Hiltner, L. Uber die Bedeutung der Wurzelknollchen von Alnus glutinosa
fur die Stickstoffernahrung dieser Pflanze. Landw. Vers. Sta. 46: 153-161. 1896.

170 Kellerman, K. F. Nitrogen-gathering plants. Yearb. Dept. Agr. U. S. A.,
1910, 213-218.

171 Bottomley, W. B. The root nodules of Ceanothus americanus. Ann. Bot.
29: 605-610. 1915.

172 Arzberger, E. G. The fungous root-tubercles of Ceanothus americanus,
Elaeagnus argentea, and Myrica cerifera. Mo. Bot. Gard. 21 Ann. Rpt. : 60-103,
1910.

173 Shibata, K., and Tahara, M. Studien uber die Wurzelknollchen. Bot.
Mag. Tokyo, 31: 157-182. 1917.

BACTERIA FIXING ATMOSPHERIC NITROGEN 139

strated. 174 Burrill and Hansen 175 came to the conclusion that the
root-nodules of Ceanothus (C. americanus), Alnus, Cycas revoluta, and
Myrica are not caused by Bad. radicicola. The evidence that Elaeagnus
and Podocarpus nodules are caused by B. radicicola is not conclusive.
It is still questionable whether nitrogen fixation by any of these plants
takes place, 176 although it is claimed 176 * that some plants (like Casua-
rina) are thus able to grow readily in very poor sandy soil. The ques-
tion of symbiosis with fungi (mycorrhiza formation) is discussed else-
where.

It is of interest to point out, in this connection, that there are legumi-
nous plants, which do not form any nodules. These include Gymno-
cladus, Carcis, Gleditsia and the Cassias of the subfamily Caesal-
pinaceae.

Nodule formation in the leaves of some plants. A condition similar to
nodule formation by bacteria on the roots of leguminous plants has been
observed on the leaves of certain tropical plants, namely the Myrsina-
ceae, such as Ardisia, certain Rubiaceae, such as Pavetta and Grumilea.
Koorders 177 demonstrated the presence of either bacteria or fungi in the
bloom bud hydathodes of nineteen species of tropical plants, repre-
senting six genera; a symbiotic relation was found to exist between the
host plant and the microorganisms. Zimmermann 178 was the first to
show that the nodules on the leaves of the Rubiaceae (four species
examined) are filled with bacteria. He also found nodules on the upper
side of the leaf of Pavetta lanceolota and on the under side of P. angusti-
folia. The bacteria were present in chains and as longer forms. P.
indica had even a greater number of nodules scattered over the whole
surface of both sides of the leaf and formed dark green spots. The
bacteria do not penetrate the cell but are found in the intra-cellular

174 Spratt, E. R. The formation and physiological significance of root nodules
in the Podocarpineae. Ann. Bot. 26: S01-814. 1912; The root nodules of the
Cycadaceae. Ibid. 29: 619-626. 1915.

178 Burrill and Hansen, 1917 (p. 126).

176 Miehe, H. Anatomische Untersuchung der Pilzsymbiose bei Casuarina
equisetifolia nebst einigen Bemerkungen liber das Mykorhizenproblem. Flora,
111-112: 431-449. 1918.

176a Rao, R. A. Casuarina root nodules and nitrogen fixation. Madras Agr.
Dept. Yearbook, 1923, 60-67.

177 Koorders, S. H. Uber die Bluthenknospen Hydathoden einiger tropischen
Pflanzen. Ann. Jard. Bot. Buitenzorg, 14: 354-477. 1897.

178 Zimmermann, A. tlber Bakterienknoten in den Blattern einiger Rubiaceen.
Jahrb. wiss. Bot. 37: 1-11. 1902.

140 PRINCIPLES OF SOIL MICROBIOLOGY

spaces. An organism belonging to the mycobacteria (My cob. rubia-
cearum) was isolated 179 from the leaf nodules. The same organism was
also isolated from Pavetta and other plants. Miehe 180 isolated a rod-
shaped organism, Bac. foliicola, active in forming nodules on the leaves
of Ardisia. It is a motile rod (1-2.5 by 0.4-0.5ju) with peritrichic fiagella
and later changes into a branching form. These "bacteroids" may be
found in the cells of the leaves and also on special media. The bacteria
are already present in the seeds, between the embryo and the endosperm,
so that the plants do not have to be inoculated anew with each new
growth. In this respect they are similar to the endotrophic mycorrhiza
of the Ericaceae which are considered elsewhere. When the young
plants grow, the bacteria follow the growing tip to the new parts of the
plant, as they develop. The bacteria are eventually found in the entire
plant, where they develop in masses in the intracellular spaces. With
the development of the fruit, the bacteria are enclosed in the embryo
sack and remain with the seed. Miehe concluded that Bac. foliicola
fixed nitrogen; he recognized this phenomenon as one of hereditary
symbiosis. The bacteria forming nodules on the leaves of Pavetta
indica and Chomelia asiatica enter the stomata of the leaf, live there and
fix the nitrogen from the air. The bacteria are found at all the life
stages of the plant, symbiosis being developed to a much greater extent
than in the Leguminosae and being hereditary in nature. Plants freed
from bacteria, by warming the seed for 25 minutes at 50°C, develop
very slowly and suffer from lack of nitrogen. The bacteria are aerobic,
rod-shaped cells. 181

The presence of nitrogen-fixing bacteria in the swollen glands on the
points of the leaves of Dioscorea macroura has also been demonstrated. 182

179 von Faber, F. C. Die Bakteriensymbiose der Rubiaceen. Jahrb. wiss.
Bot. 54: 243-264. 1914; also Ibid. 51: 285-295. 1912.

180 Miehe, H. Weitere Untersuchungen liber die Bakteriensymbiose bei
Ardisia crispa. Jahrb. wiss. Bot. 53: 1-54. 1913; 58: 29. 1917. Ber. Bot.
Gesell. 29: 156. 1911; 34: 576. 1916.

181 Rao, K. A. A preliminary account of symbiotic nitrogen fixation in non-
leguminous plants, with special reference to Chomelia asiatica. Agr. Jour. India,
18: 132-143. 1923.

182 Orr, M. V. Nitrogen fixation in leaf glands. Notes from the Roy. Bot.
Gard., Edinburgh, 14: 57-72. 1924.

CHAPTER V

Heterotrophic, Aerobic Bacteria Requiring Combined Nitrogen

General classification. The heterotrophic bacteria requiring combined
nitrogen comprise the large numbers of organisms developing on the
common plate used for counting bacteria and probably a still greater
number of organisms, which develop very slowly or do not develop upon
the plate at all. Morphologically they consist of spore-forming and
non-spore forming rods, cocci and spirilli. Physiologically they take
part in numerous soil processes, especially in the decomposition of
both simple and complex organic substances in the soil including pro-
teins, their derivatives, and other nitrogen compounds ; celluloses, pen-
tosans, and other complex and simple carbohydrates; fats and various
other ingredients of natural organic matter. Morphology alone is an
insufficient basis for the classification of these organisms. Just as in
the general classification, one must consider the various physiological
processes in which these bacteria are concerned. The system used
here is far from satisfactory, due to insufficient knowledge concerning
the organisms themselves. This system is bound to change with the
advance of our knowledge.

The cellulose-decomposing bacteria, the nitrate and sulfate-reducing
bacteria, and the urea-decomposing organisms are treated separately,
because of their specific physiology and the special methods which
are essential for their isolation, cultivation and study. Some of these,
especially some of the urea-decomposing forms and nitrate-reducing
bacteria, are no doubt modifications of the more general groups consid-
ered here. As a general basis of classification, the following one may
be used conveniently:

I. Aerobic bacteria:

1. Spore-forming rods

2. Non-spore forming rods

3. Cocci

4. Spirilli

II. Anaerobic bacteria

The difference between the aerobism and anaerobism of soil bacteria
is largely one of degree and not of kind, as will be shown later. The

141

142 PRINCIPLES OF SOIL MICROBIOLOGY

anaerobic bacteria, especially the obligate forms, will be treated sepa-
rately because of their peculiar physiology. Of the two groups of aero-
bic rod-shaped bacteria, the non-spore formers are more numerous than
the spore-formers. The latter usually become very active when fresh
organic matter, rich in proteins, is added to the soil but they soon
sporulate and generally remain in the soil in that condition until another
favorable period arrives. The non-spore forming bacteria and cocci,
living upon the colloidal film surrounding the inorganic soil particles,
make up the bulk of the numbers of the soil population. Most of these
organisms have not yet been described at all or only very insufficiently.
Their physiological activities are also insufficiently studied and their
role in soil processes is little understood.

Spore-forming bacteria. The spore-forming, aerobic, heterotrophic
bacteria have been studied more completely than the non-spore formers
or the anaerobic bacteria. This is due to the fact that they readily
develop on the common gelatin and agar media, forming large charac-
teristic colonies. When a short period of incubation is used, they are
found to be among the most numerous organisms developing on the
plate. Houston 1 found in 1898 four common spore-forming bacteria
in the soil: Bac. mycoides, Bac. subtilis (which was, according to Conn, 1
Bac. cereus), a "granular bacillus," equivalent to Bac. megatherium, and
Bac. mesentericus representing a group composed of a number of ill-
defined, small spore-forming organisms. 2 Houston states that Bac.
mycoides is present in the vegetative stage and as spores. Others 3
found the spore-forming bacteria to be present in the soil only in the

1 Houston, 1898 (p. 14).

2 Conn, H. J. Soil flora studies. III. Spore-forming bacteria in soil. N. Y.
Agr. Exp. Sta. Tech. Bui. 58. 1917.

3 Conn, H. J. Are spore-forming bacteria of any significance in soil under
normal conditions? N. Y. Agr. Exp. Sta. Tech. Bui. 51. 1916.

PLATE VIII

43. Bacterium phlel: A, colony of organism on washed agar containing inor-
ganic salts, with petroleum vapor as the only source of energy; B, colony on
agar with inorganic salts and 1 per cent glycerol (after Sohngen and de Rossi).

44. Deep agar colonies of anaerobic bacteria: colonies of Bac. perfringens in
nitrate glucose agar (from Weinberg and Seguin).

45. Deep agar colonies of anaerobic bacteria: Bac. pulrificus in glucose agar,
48 hours old (from Weinberg and Seguin).

46. Ash-agar plate showing the organism forming nodules on the roots of
a, Genista tincloria, 25 days old; b, Pisurn sativum, 7 days old (from Burrill and
Hansen).

PLATE VJII

43

#

0, 46

HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 143

form of spores. The spores vegetate in the presence of a large amount
of organic matter and an excess of moisture. 4

A detailed study of the spore-forming bacteria has been made by
various investigators 5-8 and recently by Ford 9 and associates, whose
work is used as a basis for the following classification.

Classification of Spore-forming Bacteria 9

Group I. Subtilis group

Small, homogeneous, sluggishly motile organisms measuring 0.4 by 1.5 to
2.5ju. No threads on glucose agar. Central or excentric spores, oval, measuring
0.5 by 0.75 to 0.88;u, often retaining terminal tags of protoplasm. Growth on
solid media hard and penetrating, with tenacious scums on fluid media.
Bacillus subtilis Cohn
Bacillus subtilis-viscosus Chester
(Characterized by viscosity)

Group II. Mesenlericus group

Small, homogeneous, actively motile organisms measuring 0.5 by 2 to 4^.
They often produce long threads on glucose agar. The spores measure 0.5 by
1 to 1.12/i, oval and retaining terminal tags of protoplasm. The growth on
solid media is a soft pultaceous mass with tendency to wrinkle; on fluid media,
growth is in the form of a friable easily-broken scum.
Bacillus vulgatus (Flugge) Trevisan

(Bacillus mesentericus vulgatus Flugge)
Bacillus mesentericus (Flugge) Migula

(Bacillus mesentericus fuscus Flugge)
Bacillus aterrimus Lehmann & Neumann

(Bacillus mesentericus niger Lunt)
Bacillus globigii Migula

(Bacillus mesentericus ruber Globig)

4 Winogradsky, 1924 (p. 542).

6 Gottheil, O. Botanische Beschreibung einiger Bodenbakterien. Centrbl.
Bakt. II, 7: 430^35, 449^65, 481^97, 529-544, 582-591, 627-637, 680-691, 717-730.
1901.

6 Neide, E. Botanische Beschreibung einiger sporenbildenden Bakterien.
Centrbl. Bkt. II, 12: 1-32, 161-176, 337-352, 539-554. 1904.

7 Chester, F. D. Observations on an important group of soil bacteria. Organ-
isms related to Bacillus subtilis. Del. Agr. Exp. Sta., Rept. 15: 42-96. 1904.
A review of the Bacillus subtilis group of bacteria. Centrbl. Bakt. II, 13: 737-
752. 1904.

8 Holzmiiller, K. Die Gruppe des Bacillus mycoides Flugge. Centrbl. Bakt.
II, 23: 304-354. 1909.

9 Ford, W. W., Lawrence, J. S., Laubach, C. A., and Rice, J. L. Studies on
aerobic spore-bearing non-pathogenic bacteria. Jour. Bact. 1: 273-320, 493-534.
1916.

144 PRINCIPLES OF SOIL MICROBIOLOGY

Bacillus niger Migula

{Bacillus lactis niger Gorini)
Bacillus mesentericus var. flavus
Bacillus panis Migula

(Bacillus mesentericus panis viscosus Vogel)

(Motility lost by capsule formation)

Group III. C ohaer ens-simplex group

Motile organisms somewhat larger than either Bacillus subtilis or Bacillus
mesentericus, measuring 0.37 to 0.75 by 0.75 to 3^. Thicker and longer forms on
glucose agar. Involution and shadow forms are common and appear early.
The spores are cylindrical, measuring 0.56 to 0.75 by 1 to 1.5^. A soft mass is
formed on solid media; turbidity with little or no scum on liquid media.
Bacillus cohaerens Gottheil
Bacillus simplex Gottheil
Bacillus agri Ford and associates
Bac. asterosporus and Bac. teres A. M. and Neide belong also to this group.

Group IV. Mycoides group

Large organisms with square ends growing in long chains. Single cells measure
0.5 by 3 to 6fi. On glucose agar, the organisms are thicker and longer and are
made up of globular bodies. Tendency for the organisms to grow in curves or
spirals. The spores are central or excentric, round or oval to cylindrical, measur-
ing 0.75 to 1 by 1 to 2 fi. Dry and penetrating growth on solid media; firm
tenacious scum on liquid media.
Bacillus mycoides Flugge
Bacillus prausnitzii Trevisan

(Bacillus ramosus liquefaciens Prausnitz)
Bacillus adhaerens Ford and associates
(No motility)

Group V. Cereus group

Large, motile organisms with rounded ends, measuring 0.75 by 2.25 to 4fi. Tend
to grow in short chains. Thicker and longer on glucose agar, where protoplasm
is converted into globular bodies. Central or excentric spores, cylindrical, meas-
uring 0.5 to 0.75 by 1.12 to 1.5/x. Spores retain protoplasm at one or both ends,
often resembling enlarged subtilis or mesentericus spores. A soft pultaceous mass
is formed on solid media, with tendency to fold or wrinkle; thick friable scum on
liquid media.

Bacillus cereus Frankland (The Bac. ellenbachensis often referred to as an
important soil organism belongs here).

Bacillus albolactus Migula

Bacillus cereus var. fluorescens Ford and associates

Group VI. Megatherium group

Very large, actively motile organisms, measuring 0.75 to 1.25 by 3 to 9p. Long
forms are often produced ; these spread out, lose their cytoplasm and show peculiar

HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 145

aggregations of protoplasm at the periphery. The protoplasm is rapidly con-
verted into peculiar globular, highly refractile bodies, particularly on glucose
agar. Shadow and transparent forms appear early. The spores are central,
excentric or sub-terminal, oval to cylindrical, measuring usually 0.75 to 1.12
by 1.5 to 2ju. Spores vary greatly in shape, being sometimes round, sometimes
rectangular, often reniform. Growth on solid media as thick pultaceous mass,
on liquid media as turbidity with little or no scum formation.

Bacillus megatherium De Bary

Bacillus petasites Gottheil

Bacillus ruminatus Gottheil

Group VII. Round terminal spored group

Small, actively motile organisms, measuring 0.5 to 0.75 by 1.5 to 3ju, often
forming long threads in old cultures. Protoplasm homogeneous. Spores sub-
terminal or terminal, round, thicker than the organisms from which they spring,
measuring 1 to 1.5ju in diameter.

Bacillus pseudotetanicus (Kruse) Migula

(Bacillus pseudotetanicus var. aerobius Kruse)
Bacillus fusiformis Gottheil

Group VIII. Cylindrical terminal spored group

Small, thin, actively motile organisms, measuring 0.37 to 0.5 by 2.5 to 4/z.
Slightly larger on glucose agar but no change in character of protoplasm. Spores
terminal, cylindrical, measuring usually 0.75 by 1.12 to 1.5/*.

Bacillus circulans Jordan

Bacillus brevis Migula

Bacillus terminalis Migula

Group IX. Central spored group

L^ng, actively motile organisms with pointed ends, measuring 0.37 to 0.5 by
1.12 to 4ju. Slightly larger on glucose agar, but no change in character of proto-
plasm. The spores develop in the middle of the rods, which become spindle-
shaped. The spores are large, cylindrical, measuring 0.6 to 0.8 by 1.12 to 1.5/x.

Bacillus centrosporus Ford and associates

Bacillus laterosporus Ford and associates

A summary of the characteristic points of the spore-forming bacteria,
recognized by A. Meyer and his associates is given in table 13. 10

Occurrence of aerobic, spore-forming bacteria in the soil. By the use
of gelatin plates the three most common spore-forming bacteria in the
soil can be readily recognized by speed of gelatin liquefaction and type of
colony. Except for the non-spore forming Bad. fluorescens, Bac.
mycoides is the most rapid liquefier; it produces large filamentous to
rhizoid colonies. Bac. cereus liquefies gelatin almost as rapidly as Bac.

10 Stapp, 1920 (p. 213).

146

PRINCIPLES OF SOIL MICROBIOLOGY

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HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 149

mycoides and usually forms round colonies with entire edges; the surface
membrane contains granules which tend to be arranged concentrically.
Bac. megatherium liquefies gelatin more slowly; its colonies are seldom
over 1 cm. in diameter and are characterized by a flocculent center
composed of white opaque granules, surrounded by a zone of clear
liquefied gelatin; the smaller colonies have no surrounding zone and
are recognized only by their granular structure. 11

These organisms as well as various other spore-forming bacteria are of
universal occurrence in the soil. The following forms were demon-
strated 12 in the soil of Northern Greenland: Bac. subtilis, Bac. mesen-
tericus, Bac. asterosporus, and Bac. malaberensis. According to Flugge 13
Bac. mycoides is present in almost every soil examined. Holzmiiller 14
also found one form or another of Bac. mycoides in every soil. Gottheil
found Bac. asterosporus very abundantly in the soil as well as Bac.
ellenbachensis, and to a less extent Bac. cohaerens, Bac. fusiformis, Bac.
pctasites, Bac. graveolus, Bac. pumilus, Bac. ruminatus, Bac. subtilis and
Bac. tumescens. Bac. simplex was found only once, while the presence
of Bac. mycoides and Bac. carotarum could not be established

According to Ford and associates Bac. cereus is very predominant in
Maryland soils, followed by Bac. subtilis; Bac. mesentericus was more
abundant than Bac. vulgatus, while Bac. megatherium , Bac. petasites and
Bac. mycoides were found only rarely. Five hundred and twenty cul-
tures were isolated from eight soils (five near Baltimore and three from
Nazareth, Pa.). All soil samples were boiled in water for 20 minutes
just before plating, to kill all the vegetative cells of bacteria as well as
the other soil organisms and leave only the spores of the spore-forming
bacteria. (See tabulation on p. 150.)

The three most common types of spore-forming bacteria found in
the soil by Conn were: Bac. megatherium (averaging about 375,000 per
gram), Bac. mycoides (225,000 per gram) and Bac. cereus (180,000 per
gram) .

The relative number of spore-forming bacteria in the soil depend on
the length of the incubation period. When a short incubation period
is used, nearly half of the colonies on the plate may be found to consist

11 Conn, 1917 (p. 142).

12 Barthel, Ch. Recherches bacteriologiques sur le sol et sur les matieres
fecales des animaux polaires du Groenland septentrional. Saertryk MeddeL
Groenland 64, Kopenhagen. 1922.

13 Fliigge, 1896 (p. x).

14 Holzmiiller, 1909 (p. 143).

150

PRINCIPLES OF SOIL MICROBIOLOGY

PRESENCE IN NUMBER
OF SOIL SAMPLES

ORGANISM

B. petasites

B. cereus

B. megatherium

B. subtilis

B. mesentericus

B. vulgatus

B. mycoides

B. mesentericus var. flavus
B. cereus var. fluorescens . .

B. fusiformis

B. brevis

B. simplex

B. cohaerens

B. agri

Total isolations

of spore-forming organisms. 15 When a long incubation period is used
they are found to form only about 5 per cent of the colonies. 1617 The
presence of spore-forming bacteria, which are destroyed with difficulty
by partial sterilization, varies with the soil. 18 Here belong Bac.
mycoides, Bac. mesentericus and Bac. mesentericus ruber. The simplest
way of destroying these is to heat the soil for 30 minutes at 100°C.
on seven consecutive days and then to incubate the soil, between suc-
cessive sterilizations, at room temperature.

Non-spore forming bacteria. Although the heterotrophic non- spore
forming bacteria include the most predominant group of soil organisms
developing on the plate and even more so in the microscopic examination
of the soil, very little attention has been paid to their identification.
This has been due, probably, to the fact that these organisms grow
poorly and only very slowly on laboratory culture media. The inap-
preciable results obtained by the ordinary chemical tests led to the
general assumption that they are of little importance in the soil. The
fact must also be kept in mind that certain very important physiological

15 Chester, F. D. Study of the predominating bacteria in a soil sample.
Agr. Exp. Sta., Rept. 14: 52-63. 1903.

16 Hiltner and Stormer, 1903 (p. 12).

17 Conn, 1917 (p. 142).
18 Eckelmann, 1918 (p. 641).

Del.

HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 151

groups of soil organisms, such as the Thiobacillus group of sulfur bac-
teria, the nitrite, nitrate forming and other autotrophic bacteria,
as well as various heterotrophic bacteria, such as many of the aerobic
cellulose decomposing bacteria, certain urea bacteria, etc., belong to this
group of organisms.

Except for the physiological groups just mentioned, the study of the
heterotrophic, non-spore-forming bacteria, which do or do not develop
on the common synthetic and nutrient media, has been neglected. This
has been largely a result of the lack of proper methods of study.
Organisms have been looked for which take part in the various known
processes, largely in the nitrogen transformation in the soil. If an
organism did not take part in the processes of nitrification or sulfur-
oxidation, nitrogen-fixation or cellulose decomposition, and if it did
not produce ammonia rapidly from proteins, it was assumed to be un-
important in the soil.

We must assume from the meager information available that the
non-spore-forming bacteria take part in the slow but constant decom-
position of the soil organic matter. Winogradsky considers them as the
native (autochtonous) population of the soil. When fresh organic matter
is added to the soil, the fungi and the spore-forming bacteria at once
become active, until the organic matter is reduced to a certain consist-
ency (so-called "humus"). These organisms then become inactive
again until a fresh supply of energy is introduced into the soil.
The "humus" left is constantly acted upon by the non-spore-forming
bacteria (and actinomyces), which mineralize it, liberating the nitro-
gen and other mineral elements into available forms.

Classification. The heterotrophic, non-spore-forming aerobic bac-
teria usually produce punctiform colonies on agar and gelatin, are
chromogenic or non-chromogenic, motile or non-motile, some liquefy
gelatin rapidly, while others only slowly or not at all. The non-spore-
forming bacteria, which form the most abundant group of soil organisms,
as shown both by microscopic and cultural methods, can be divided into
(1) the forms that liquefy gelatin rapidly and (2) the forms that liquefy
gelatin slowly.

The most important representatives of the group of rapid liquefying
organisms is the Bad. fluorescens or Pseud, fluorescens. The whole
group is often spoken of as the fluorescens group, although many of the
organisms never produce any fluorescens.

The representatives of the second group comprising the bacteria
which form pin-point colonies on the agar and gelatin plate and which

152 PRINCIPLES OF SOIL MICROBIOLOGY

liquefy gelatin only very slowly, are characterized by poor growth in all
liquid media and by the formation of punctiform colonies on gelatin.
These organisms are usually less than one micron in length and less
than half a micron in diameter, being short rods or cocci; growth on
agar streaks is fair, soft, smooth, glistening, slimy to watery. Due to
the fact that they grow only poorly in liquid media, they do not lend
themselves readily to physiological studies. They also appear much
the same morphologically. The silica gel plate with soil extract as the
nutritive ingredient presents a new and promising method for their
study.

Conn 19 divided these organisms into five groups on the basis of their
growth upon a synthetic medium (1.0 gram NH 4 H 2 P0 4 , 0.2 gram
KC1, 0.2 gram MgS0 4 , 10.0 sugar in 1000 cc. of water, adjusted to
pH 7.0):

1. Organisms forming small short rods, usually under 0.5 mi-
cron in diameter, non-motile or having one or possibly two polar fla-
gella; no tendency to change in morphology but very variable in their
physiology.

2. Organisms that appear for a day or two after inoculation on a
new medium as small short rods, less than 0.5^ in diameter, then shorten
and appear like micrococci. All liquefy gelatin, slowly however, and
can be separated on the basis of physiological characteristics.

3. Small short rods, with a tendency to produce long filaments, usually
unbranched, but frequently branched.

4. Organisms consisting, in young cultures, mostly of branching forms,
apparently produced by the germination of small spherical arthrospores.
The branching forms disappear in a few days, leaving the coccoid forms.

5. Organisms occurring normally as cocci, but with a tendency to
produce rods and filaments after a few days of growth on ordinary
media. This group is more abundant in manure than in soil.

The first two groups are most numerous in the soil. According to
Winogradsky 20 the cocci predominate, as shown by the direct micro-
scopic method (including probably also the short rods). These organ-
isms were found to grow in groups (masses, zooglea) imbedded in the
colloids which cover the soil particles.

19 Conn, H. J. Soil flora studies. VI. The punctiform-colony-forming bac-
teria in the soil. N. Y Agr. ^xp. Sta. Tech. Bui. 115. 1925.

20 Winogradsky, 1925 (p. 7).

HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 153

The term "Micrococcus" was applied 21 to designate the genus of
irregular mass-forming cocci. Sixteen species were separated on the
basis of gelatin liquefaction, nitrate reduction and ability to utilize
certain ammonium salts as the only source of nitrogen.

The fact must also be mentioned that a few spore-forming organisms
are found to form punctiform colonies. They are not related to the
other spore-forming bacteria in their general physiology and stand also
apart from the "slow growers." Except the Bad. fluorescens and
Bad. caudatum, the other non-spore-forming bacteria cannot be readily
recognized by their colonies on gelatin or agar. The rapid liquefaction
of the gelatin by the former and the orange color of the latter allow a
ready recognition of these two types. Bad. aerogenes and Bad. coli
isolated from the soil can be readily distinguished from Bad. coli of
fecal origin by the fact that the former use citrates as a source of carbon
and the latter do not; 22 also by the production of a red iron rust growth
on Harder's medium. 23

Occurrence of non-spore-forming bacteria in the soil. The non-spore-
forming bacteria are numerically the largest group of soil microorganisms.
It still remains to be demonstrated, however, what bulk they occupy in
the soil population and what relative importance may be ascribed to
them in transformations in the soil. Seventy-five per cent of the total
number of colonies developing on the plate are non-spore-forming bac-
teria and cocci, the other 25 per cent include the spore-forming organ-
isms and actinomyces. The seventy-five per cent of the colonies is made
up largely of the slow growing organisms; between 2 to 60 millions of
these organisms, as determined by the plate method, are found per 1
gram of soil. The non-spore-forming bacteria are believed to be very
active in the soil (Conn, Winogradsky) , particularly in view of their
great variability as affected by the soil treatment. Conn found that
a certain soil contained 350,000 rapid-liquefying colonies, 11,000,000
punctiform colonies and 4,700,000 spore-formers and actinomyces, be-
fore aeration; after aeration for one day these numbers changed to

21 Hucker, G. J. Studies on the coccaceae. I. Previous taxonomic studies
concerning the genera of the coccaceae. N. Y. Agr. Exp. Sta. Tech. Bui. 99.
1924; II. A study of the general characters of the micrococci. Ibid. 100. 1924;
III. The nitrogen metabolism of the micrococci. Ibid. 101. 1924; IV. The
classification of the genus micrococcus Cohn. Ibid. 102, 1924.

22 Koser, S. A. Utilization of the salts of organic acids by the colon-aerogenes
group. Jour. Bact. 8: 493-520. 1923;9:59-77. 1924.

23 Murray, T. J., and Skinner, C. E. Differentiation of B. aerogenes and B. coli
of fecal origin. Proc. Soc. Exp. Biol. Med. 23: 104-106. 1925.

154 PRINCIPLES OF SOIL MICROBIOLOGY

1,500,000 of the first, 22,500,000 of the second, and 4,000,000 of the
third. Aeration of soil and the addition of organic matter bring about
the greatest numerical increase in the group of non-spore-forming bac-
teria in the soil. The above numbers as determined by the plate method
represent only a fraction of the actual abundance of these organisms in
the soil, since they live in zooglea-like masses imbedded in the soil col-
loids and cannot be readily separated into individual cells.

Of the non-spore-forming bacteria and cocci, some are especially
abundant in the soil. The available information on this subject is very
meager due largely to the difficulty of studying some of the slow growing
organisms on artificial media. The Bad. fluorescens is especially abun-
dant, having been found in soils all over the world. The same is true of
certain cocci or very short rods.

Severin 24 found rods to predominate in freshly plowed manured soil
as well as in manure itself; however, in two weeks cocci predominated
According to Houston, 25 Bad. vulgar e (Proteus vulgaris), Bad. prodigi-
osum and various Saicinae are less abundant in the soil than the
spore-forming bacteria, actinomyces, and the Bad. fluorescens. When
inoculated into soil, Bad. prodigiosum persisted only when the soil
was previously sterilized and kept moist; when the soil was air dried
or when the inoculation took place into non-sterile soil, the organism
was rapidly destroyed. The occurrence of brown fluorescent bacteria
in the soil was pointed out by Bazarewski. 26 Barthel 27 found in a
North Greenland soil Bad. fluorescens, Bad. caudatum, Bad. pundatum,
Bad. violaceum, Bad. ladis viscosum, Bad. umbilicatum, Bad. ochraceum
and Bad. zopfii. Among the cocci, Barthel found Tetracoccus and Sar-
cina flava. In addition to these organisms, various other bacteria,
like those forming a blue pigment, those accompanying the nitrogen-
fixing organisms, various spirilli, vibrios, and cocci have been reported
to be found in the soil by different investigators. Certain organisms,
like Bad. vulgare, Bad. colt and others, present abundantly in manure and
feces, are thus abundantly introduced into the soil and may survive
there for a long time, some of which, such as various proteolytic and
cellulose-decomposing bacteria, may even become active there.

24 Severin, S. A. Die im Miste vorkommenden Bakterien und deren physi-
ologische Rolle bei der Zersetzung desselben. Centrbl. Bakt. II, 1: 100-104.
1895; Zhur. Opit. Agron. 1: 463-489. 1900.

"Houston, 1898 (p. 14).

26 Bazarewski, S. v. tlber zwei neue farbstoffbildenden Bakterien. Centrbl.
Bakt. II, 15: 1-7. 1905.

27 Barthel, 1922 (p. 149).

HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 155

The bacteria of the so-called colon group found abundantly in the
feces of warm blooded animals differ, in certain cultural characteris-
tics, 28 from those found in the soil, the former being largely Bad. coli
and the latter belonging to the Bad. aerogenes group, which is even cap-
able of fixing small amounts of nitrogen. Out of 467 strains of bacteria
isolated from various soils by Chen and Rettger, 430 were identified as
Bad. aerogenes, 17 as Bad. cloacae and only 20 as Bad. coli; the sources
of the Bad. coli strain were shown by a sanitary survey to be not entirely
free from animal pollution. However, all of 173 organisms found in the
feces of various animals were typical Bad. coli. When Bad. coli and
even Bad. aerogenes are added to the soil in considerable numbers, they
rapidly die out, 29 since conditions are not very favorable for their
development, although Bad. coli was found 30 to survive in the soil for 4
years.

It is of interest to note here that Stoklasa 31 found the following
organisms in the "rhizosphere" or in close proximity to the root
system of plants, consisting largely of non-spore-forming bacteria:

Bad. acidi laclici a and /3

Bad. pneumoniae

Bad. coli group

Bad. proteus vulgaris

Bad. ochraceum

Bad. fulvum

Bad. pundatum

Bad. putidum

Bad. fluorescens liquefaciens

Bad. anthracoides
Azotobader chroococcum
Butyric acid bacteria
Bac. mycoides
Bac. mesentericus
Mycoderma

Cladosporium herbarum
Fusarium

Thermophilic bacteria. It has been known since the work of Globig, 32

28 Chen, C. C., and Rettger, L. F. A correlation study of the colon-aerogenes
group of bacteria, with special reference to the organisms occurring in the soil.
Jour. Bact. 5: 253-298. 1920; Levine, M. Bacteria fermenting lactose and their
significance in water analysis. Iowa Engin. Exp. Sta. Bui. 62. 1921.

29 Skinner, C. E., and Murray, T. J. The viability of B. coli and B. aerogenes
in soil. Jour. Inf. Dis. 38: 37-41. 1926.

30 Young, C. C, and Greenfield, M. Observation on the variability of the
Bacterium coli group under natural and artificial conditions. Amer. Jour. Publ.
Health, 13: 270-273. 1923. See also Koser, S. A. Coli-aerogenes group in
soil. Jour. Amer. AYater Works Assn. 15: 641-646. 1926.

"Stoklasa, 1926 (p. xiii).

"Globig. Uber Bakterienwachstum bei 50-70°. Ztschr. Hyg. 3:294-321.

156 PRINCIPLES OF SOIL MICROBIOLOGY

Macfayden and Blaxall 33 in 1888, and other workers soon following 34 ' 35
that there are bacteria in the soil which are capable of growing at high
temperatures, namely at 50° to 70°C, and which refuse to grow in the
laboratory at room temperature. Various investigators 36 " 38 found in
stable manure organisms capable of growing at temperatures up 79.5°C.
Schloesing 39 demonstrated in 1892 that the self-heating and burning of
hay, cotton, manure, etc., is caused by microorganisms which he called
thermogenic bacteria. It may be here largely a question of thermo-
tolerant organisms rather than strict thermophiles. 40 Thermophilic
bacteria were found in sands of the Sahara Desert, 41 but are absent in
forest soils. The distribution of these organisms in the soil seems to
depend on the manure used. Garden soils heavily manured may con-
tain 1 to 10 per cent of the flora developing on the plate as thermophilic
forms, while field soils contain only 0.25 to per cent. Uncultivated

33 Macfayden, A., and Blaxall, F. R. Thermophilic bacteria. Jour. Path.
Bact. 3: 87-99. 1894.

34 Rabinowitsch, L. "fiber die thermophilen Bakterien. Ztschr. Hyg. 20:
154. 1895. Oprescu, V. Studien iiber thermophile Bakterien. Arch. Hyg.
33: 164. 1898. Schillinger, A. Uber die thermophilen Bakterien. Hyg.
Rundsch. 1898, p. 568. Tsiklinsky, P. On microorganisms living at high tem-
peratures. Russ. Arch. Pathol. 5, 1898 (Ambroz, A. tlber das Phanomen der
Thermobiose bei den Mikroorganismen. Centrbl. Bakt. I, Ref. 48: 257-279,
289-312. 1910). Sames, T. Zur Kenntnis der bei hoheren Temperaturen wach-
senden Bakterien und Streptothrixarten. Ztschr. Hyg. 33: 313. 1910.

35 Blau, O. Ueber die Temperaturmaxima der Sporenkeimung und der Sporen-
bildung, sowie die supramaximalen Totungszeiten der Sporen der Bakterien,
auch derjenigen mit hohen Temperaturminima. Centrbl. Bakt. II, 15: 97-143.
1906.

36 Burrill, T. J. The biology of silage. 111. Agr. Exp. Sta. Bui. 7. 1889.

37 Schloesing, Th. Sur la fermentation formenique du fumier. Compt. Rend.
Acad. Sci. 109: 835-840. 1889.

38 Dupont, C. Sur les fermentations aerobies du fumier de ferme. Ann.
Agron. 28: 289. 1902.

39 Schloesing, T. Contribution a l'etude des fermentations du fumier. Ann.
Agron. 18: 5. 1892.

40 Miehe, H. Die Selbsterhitzung des Heus. G. Fischer. Jena 1907.
Schiitze, H. Beitrage zur Kenntnis der thermophilen Actinomyceten und ihrer
Sporenbildung. Arch. Hyg. 67: 35-56. 1908.

41 Negre, L. Bacteries thermophiles des sables du Sahara. Compt. Rend.
Soc. Biol. 74: 814-816. 1913.

HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 157

soils may be entirely free from thermophilic organisms; 42 the geographic
condition has no influence. 43

Globig 32 isolated a number of thermophilic bacteria from the soil
and believed that, because most of them were isolated from the surface
layers, the rays of the sun supply the heat necessary to obtain the high
temperatures. Rabinowitsch 34 isolated eight species of thermophilic
bacteria from soil and feces, var} r ing in morphology (rod shaped to
comma shaped), size of spores, color of colony on agar and potato. A
number of other bacteria were isolated 35 which had an optimum at
60° to 65°C. and were killed by heating at 100°C. for 8 to 20 hours.
A variety of B. coli and a spore-bearing organism, Bac. calfactor, as well
as several fungi and actinomyces, were found capable of growing at high
temperatures and taking an active part in the decomposition of hay. 40
The size of Bac. caljacior changes with temperature at which it is grown
(on hay infusion agar at 70°C. it is 5 by 0.4/z; at 56°C. — 5 by 0.7^; at
30°C— 3 by 0.8/x). The rods occur singly, not in chains, and are motile
at favorable temperatures (above 30°). The spores are 1.5 by 0.8/x.
Spore germination takes place in 60 minutes at 60°C. ; in 85 minutes,
2 cells are already formed. This rapid multiplication accounts for the
fact that at 50°C, turbidity is definite in liquid decoctions within six
hours and an abundant growth is produced on agar.

de Kruyff 44 isolated from the soil ten species of rod shaped, thermo-
philic bacteria, most of them being long rods, forming oval to round
spores; most of them produce proteolytic enzymes and do not grow on
potato. He suggested that with a rise in temperature in tropical soil
they take the place of the ordinary bacteria. It has been established
that there occur in soils thermophilic nitrogen-fixing bacteria, 45 ther-
mophilic cellulose decomposing bacteria 46-48 and thermophilic denitri-

42 Migula, W. tiber die Tatigkeit der Bakterien im Waldboden. Forst-
wissensch/Zentrbl. 57 (Neue Folge 35): 161-169. 1913.

43 Mischustin, E. Untersuchungen xiber die Temperaturbedingungen fur
bakterielle Prozesse im Boden in Verbindung mit der Anpassungsfahigkeit der
Bakterien an das Klima. Centrbl. Bakt. II, 68: 328-344. 1926.

44 de Kruyff, E. Les bacteries thermophiles dans les Tropiques. Centrbl.
Bakt. II, 26: 65-74. 1910.

46 Pringsheim, 1911 (p. 121).

46 Pringsheim, 1913 (p. 202).

47 Kroulik, A. tiber thermophile Zellulosevergarer. Centrbl. Bakt. II, 36:
339-346. 1912.

45 Viljoen, Fred and Peterson, 1926 (p. 202).

158 PRINCIPLES OF SOIL MICROBIOLOGY

fying bacteria. 49 The nature of the medium was found 50 to have an
important influence upon the temperature optimum of the organism.
Two per cent of glucose is added to soil either alone or with 2 per
cent CaC0 3 ; this is then steamed at 100° for 20 minutes, to kill the
non-spore-forming organisms. Water is added to bring the moisture
to 16 per cent and the soils are incubated at 52°C. for 4 to 6 days, at
which time the bacteria are isolated. These bacteria grow in media
at 52°C, but not at 28° to 30°; when reinoculated into soil, they may
grow well even at 15° to 20° although not so abundantly. Miehe
assumed that these organisms can develop only in manure heaps where
a great deal of heat is generated. However, others 50 suggested that
they do not always lead a latent life in the soil, but find in summer a
suitable temperature for their development. 51 Among the activities
of the thermophilic bacteria, the decomposition of organic matter,
especially the decomposition of celluloses in manure, occupies the first
place. Many of the bacteria are strongly proteolytic. Most of them
are strictly aerobic and form spores. Some organisms are facultative
thermophilic, since they can grow at 20°, have their optimum at 50°C.
and maximum at about G0°C. 52

Mycobacteria. The Mycobacteria are a group of organisms which
differ from true bacteria by the formation of more or less long mono-
podially branched threads under normal conditions of growth. They are
largely acid fast and represent botanically a well defined group of
organisms, standing midway between the true bacteria and the actino-
myces group, which should be already more properly classified with
the fungi. A number of these organisms were isolated from manure 53
and from soil. 5455 A selective development of these organisms will
take place upon agar plates, containing the necessary minerals, an

49 Ambroz, A. Dinilrobacterium thermophilum spec, nova, ein Beitrag zur
Biologie der thermophilen Bakterien. Centrbl. Bakt. II, 37: 3-16. 1913.

60 Koch, A., and Hoffmann, C. Uber die Verschiedenheit der Temperaturan-
spruche thermophiler Bakterien im Boden und in kunstlichen Nahrsubstraten.
Centrbl. Bakt. II, 31: 433^36. 1911.

51 Krohn, V. Studien liber thermophile Schizomyceten. Ann. Acad. Sci.
Fennicae, Ser. A. 21: 1-125. 1923. Helsingfors.

52 Bergey, D. H. Thermophilic bacteria. Jour. Bact. 4: 301-306. 1919.

63 Moeller, A. Ein neuer saure- und alkoholfester Bacillus aus der Tuberkel-
bacillengruppe, welcher echte Verzweigungsformen bildet. Centrbl. Bakt. 25:
369-373. 1899.

" Sohngen, 1913 (p. 204).

" Vierling, K. Morphologische und physiologische Untersuchungen uber
bodenbewohnende Mykobakterien. Diss. Univ. Heidelberg. 1921.

PLATE IX

^t

5**.

&>

47

47. Life cycle of Azotobacter chroococcum: a, formation of symplasm by
regenerative bodies on potato, in 9 days; b, regenerative units starting to grow,
on beef gelatin, in 4 weeks; c, regenerative bodies growing from symplasm, on
beef agar, in 4 weeks; d, formation of new cells by agglomeration of regenerative
units on mannite soil extract, in 4 days; X 600 (from Lohnis and Smith).

48. Influence of composition of medium upon the morphology of Bact.
■pneumoniae: A, on beef agar, 1 day at 37°C; B, on egg agar, 1 day at 19°C; C, on
starch agar, 1 day at 19°C; X 660 (from Scales).

49. Some typical soil bacteria, as shown by the India Ink method" A, short
non-spore forming rods (bacteria); B, long, non-spore forming rods (bacteria);
C, spore-forming rods (bacilli) (from Kursteiner).

HETEROTROPHIC BACTERIA REQUIRING COMBINED NITROGEN 159

inorganic source of nitrogen and inoculated with a soil suspension,
if the cultures are placed under a bell jar together with a dish of
benzene or petroleum, and incubated at 30°C. The final isolation
and purification of the organisms can be accomplished by means of
ordinary bacteriological methods. Spore formation takes place by
contraction of the cell contents, in a manner similar to that of the
actinomyces (p. 294), giving coccus-like fragments. The colonies on
solid substrates have a certain thread-like structure (No. 43, PI. VIII).

The organisms readily utilize various hydrocarbons as sources of
energy. 54 They do not form any ammonia from proteins; 53 most of them
reduce nitrates to nitrites (similar to actinomyces). Their role in the
soil seems to consist largely in the decomposition of certain organic
compounds.

Myxobacteria. Mycobacteria occur abundantly in manure and
probably take a part in the decomposition of certain constituents of
natural organic materials. To demonstrate the presence of Myxo-
bacteria in the soil, balls of rabbit manure, previously moistened with
water and sterilized in the autoclave, are placed on the surface of the
particular layer of soil. Frequently 7-10 species are thus obtained
from one soil sample. 56

5S Krzemieniewsky, H. and S. Die Myxobakterien von Polen. Acta Soc. Bot.
Poloniae. 4: 1-54. 1926.

CHAPTER VI

Anaerobic Bacteria

Oxygen tension in the growth of bacteria. Pasteur 1 was the first to
demonstrate that there are organisms, among them yeasts, which can
live in the presence of only small traces of oxygen. Since the growth of
the microorganisms is so abundant that the small amount of oxygen
present is rapidly used up, it can be assumed that the greater part of
their development takes place in the absence of free oxygen. Pasteur
has further shown that, in the case of yeasts, growth in the absence of
oxygen takes place only in the presence of sugar utilizable by these
organisms. Those organisms which are able to grow both in the pres-

1 Pasteur, L. Animalcules infusoires vivant sans gaz oxygene libre et deter-
minant des fermentations. Compt. Rend. Acad. Sci. 52: 360. 1861; Experi-
ences et vues nouvelles sur la nature des fermentations. Ibid., 1260; 56 : 416, 1189-
1863; 75: 784. 1872; 80: 1875.

PLATE X
Heterotrophic Aerobic and Anaerobic Bacteria

50. Bac. mycoides, X 660 (from Conn).

51. Bac. cereus, X 660 (from Conn).

52. Bac. megatherium, X 660 (from Conn).

53. Bac. simplex, X 660 (from Conn).

54. Bad. vulgare, X 660 (after Omeliansky).

55. Bad. pyocyaneum, X 660 (after Omeliansky).

56. Bad. fluorescens, X 600 (after de Rossi).

57. Bac. butyricus: a, non-spore forming; b, spore forming, X 660 (from
Omeliansky).

58. Bac. sporogenes: a, 24 hour culture upon glucose bouillon; b, flagella, stained
by Loeffler's method (from Weinberg and Seguin).

59. Bac. pulrificus, 48 hour old colony in deep glucose agar (from Weinberg and
Seguin).

60. Bac.probatus: A, non-sporulating bacilli of a fresh agar culture; B, sporu-
lating bacilli of an agar culture 4-8 days old; C, spores with adhering membrane
of a 2 to 3 week old culture upon potato, X 1300 (after Viehoever and de Rossi).

61. Sarcina ureae, X 660 (after Omeliansky).

62. Bac. nitroxus, 3-day old culture, grown at 30°, X 480 (after Beijerinck and
Minkmann and de Rossi).

63. Spirillum desulfuricans, X 660 (after Beijerinck and Omeliansky).

160

PLATE X

o fl o o o G
50

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d =tccooo

11

«•*

<«■ G0000J0

52

""' ^""T*

53

51

V \ V\J '

r i * \

54

55

/ A-
-/ v /

56

/

*-,/ ^ ^

5S

59

57

,

7

<?>„= '

6Z

60

v / I

ANAEROBIC BACTERIA 161

ence and absence of free oxygen were termed by Liborius 2 "facultative
anaerobes." It has also been observed by Pasteur that certain butyric
acid bacteria grow abundantly in the liquid medium, through which a
current of carbon dioxide is passed, but are destroyed, when a current
of air is passed for 2 hours through the liquid. Those organisms, which
are unable to thrive under partial oxygen pressure and cannot withstand
even small amounts of oxygen, were termed by Liborius "obligate an-
aerobes." Beijerinck 3 divided the bacteria into two groups, according
to their oxygen need: (1) "aerophile," or those requiring a high oxygen
tension, including the aerobes and facultative anaerobes, which can
grow in ordinary atmosphere; and (2) "microaerophile," or those organ-
isms that require a more or less low oxygen tension and do not grow
readily in ordinary atmosphere. The influence of oxygen on some bac-
teria was illustrated by the accumulation of the cells in a hanging drop
preparation; the aerophiles gathered in the outer zone, while the micro-
aerophiles massed together where the oxygen tension was least. The
spirillum type was intermediate. Burri 4 could not agree with this divi-
sion and suggested that the terminology of Liborius is much more
appropriate. Not only obligate anaerobic bacteria, but also the facul-
tative forms were able to live in the complete absence of oxygen for a
number of generations without being injured.

No general minimum oxygen tension could be found for all obligate
anaerobic bacteria, but the various anaerobic forms varied in the limit of
this tension : 5 the oxygen limit for the blackleg bacillus (Bac. chauvoei)
is 1.04 per cent oxygen in the atmosphere, 0.65 per cent for Bac. tetani,
0.27 per cent for Clostridium butyricum and 0.13 per cent for Bactridium
butyricum; the obligate anaerobic bacteria could be so adapted as to
withstand some amounts of oxygen. Even many species which usually
grow in the complete absence of oxygen, such as Bac. amylobacter, can
thrive in the presence of oxygen. A typical obligate anaerobe has no
minimum oxygen tension limit, it is characterized by the existence of a
very low maximum oxygen tension and it can grow in the total absence

2 Liborius, P. Beitrage zur Kenntnis des Sauerstoffbediirfnisses der Bakterien.
Ztschr. Hyg. 1: 115. 1886.

3 Beijerinck, M. W. Ueber Atmungsfiguren beweglicher Bakterien. Centrbl.
Bakt. 14: 827-845. 1893; also Arch. Neerland., Ser. II, 2: 397. 1899; Pheno-
menes de reduction produits par les microbes. Ibid. 9: 131. 1904.

4 Burri. R. Intramolekulare Atmung, Anaerobiose und Mikroaerophilie.
Centrbl. Bakt., II, 17: 804. 1907.

5 Chudiakow, N. Zur Lehre von der Anaerobiose. Moskau. 1896 (Centrbl.
Bakt. II, 4: 389-394. 1898).

162

PRINCIPLES OF SOIL MICROBIOLOGY

of oxygen. We do not know of any true anaerobes which grow only in
the complete absence of oxygen. Small quantities of free oxygen will
even act as stimuli to obligate anaerobes.

The oxygen need of an organism was characterized 6 by the "cardinal
points" for growth and spore formation, namely: minimum, optimum,
and maximum, so that there is a gradual transition between aerobes and
anaerobes. The following cardinal points for spore formation charac-
terize a series of typical bacteria, atmospheric air at 18° and 750 mm.
pressure containing 276 mgm. of oxygen per liter:

Bac. amylobacter.
Bac. asterosporus
Bac. fusiformis. .

Bac. mycoides

Bac. simplex

Bac. subtilis

Bac. lactis

MINIMUM

OPTIMUM

MAXIMUM

mgm.

mgm.

mgm.

10 (?)

About, 25

100

5,600

6.8

70

1,061

4.3

70

1,336

6.8

276

1,263

4.3

400

4,317

20.0

400

1,336

A high maximum does not necessarily correspond to a high minimum.
The first generation of anaerobes is more sensitive to oxygen than the
following generations, which may even thrive better in the presence of a
limited oxygen supply than in its complete absence. 7 This points to
adaptation in course of time. Even in the case of a single generation,
the organism can withstand greater concentrations of oxygen after the
growth of the culture has somewhat advanced than in the beginning.
It has been claimed 8 that the growth of even obligate anaerobic bacteria

6 Meyer, A. Apparat fur die Kultur von anaeroben Bakterien und f tir die
Bestimmung der Sauerstoffminima fur Keimung, Wachstum und Sporenbildung
der Bakterienspecies. Centrbl. Bakt., II, 15: 337. 1906. Bemerkungen uber
Aerobiose und Anaerobiose. Centrbl. Bakt. I, 49: 305 316. 1909; also Ibid. II,
15: 1905; 16: 386, 481-488, 577-588, 673-687. 1906. Wund, M. Feststellung der
Kardinalpunkte der Sauerstoffkonzentration. Centrbl. Bakt. 42: 97-101, 193-
202, 289-296, 385-393. 1906.

7 Burri, 1907 (p. 161); Kiirsteiner, J. Beitrage zur Untersuchungstechnik
obligat anaerober Bakterien, sowie zur Lehre von der Anaerobiose iiberhaupt.
Centrbl. Bakt. II, 19: 1-26, 97-115, 202-220, 385-399. 1907; Burri, R. and Kiir-
steiner, J. Ein experimentaler Beitrag zur Kenntnis der Bedeutung des Sauer-
stoffentzugs f tir die Entwicklung obligat anaerober Bakterien. Ibid. 21: 289-
307. 1908; Landw. Jahrb. d. Schweiz. 1909, 422.

8 Fermi, C, and Bassu, E. Untersuchungen iiber die Anaerobiosis. Centrbl.
Bakt. I, 35: 563-568, 714-722. 1905; 38: 138-145, 241-248, 369-380. 1905.

ANAEROBIC BACTERIA 163

is greatly injured in the complete absence of oxygen; however, Kiir-
steiner 7 demonstrated that both obligate and facultative anaerobes will
thrive well for a number of generations in atmospheres free from oxygen.
Free oxygen exerts an injurious effect upon obligate anaerobic bacteria,
as pointed out already by Pasteur, the degree of injury depending on
temperature, age and abundance of cells. 9 In the following pages, the
term "anaerobe" will be applied only to the so-called "obligate
anaerobes."

The presence of suspended particles, especially in case of colloidal
suspensions, favors the growth of anaerobic bacteria possibly through
their oxygen absorption. 10

The more recent studies on oxidation-reduction processes in the
growth of microorganisms have brought out the fact that only
those bacteria are capable of growing anaerobically, which are cap-
able of activating some constituent of the medium as a hydrogen
acceptor. Some bacteria, like B. vulgar e, can activate nitrate and can,
therefore, grow anaerobically in the presence of nitrate and certain
hydrogen donators ; Bad. coli and Bad. prodigiosum can activate nitrate,
fumarate, malate and aspartate and can grow anaerobically, in the
presence of any of these substances, and with glycerol as a hydrogen
donator. 11 Recent important contributions point to the lack of cata-
lase formation by anaerobic bacteria. 12 Peroxides are formed in the
aerobic growth of bacteria and these peroxides would become injurious
to the organisms if not for the catalase which is formed and which
rapidly breaks up the peroxide into inactive oxygen and water. The
anaerobic bacteria, which] are unable to form catalase are thus subject
to the destructive action of the peroxide when grown under aerobic
conditions.

A number of indicators are employed for measuring anaerobiosis or

9 Bachmann, 1912 (p. 164).

10 Hata, S. Uber eine einfache Methode zur aerobischen Kulti vie rung der
Anaeroben mit besonderer Beriicksichtigung ihrer Toxinproduktion. Centrbl.
Bakt. I, 46: 539-554. 1908; v. Lennep, R. Folia Microb. 1: No. 3. 1913.

11 Quastel, 1925 (p. 469).

12 McLeod, J. M., and Gordon, J. Catalase production and sensitiveness to
H 2 2 among bacteria; with a scheme of classification based on these properties.
Jour. Path. Bact. 26: 326-331, 332-343. 1923; The relation between the reducing
powers of bacteria and their capacity for forming peroxide. Ibid. 28: 155-164,
147-153. 1925.

164 PRINCIPLES OF SOIL MICROBIOLOGY

determining the end point of free oxygen. 13 However, various difficul-
ties are found in an attempt to use indicators, such as methylene blue,
as criteria in anaerobiosis.

For the existence of even obligate anaerobes in the soil we need not
imagine a soil atmosphere free from atmospheric oxygen, but simply
that anaerobic conditions, favorable for the activities of these organisms,
are produced due to the active utilization of the oxygen and production
of CO2 by aerobic organisms, which results in a reduction of the oxygen
tension. This can be imitated artificially in the laboratory, when
anaerobes are grown readily under ordinary conditions, in the pres-
ence of rapidly growing aerobic bacteria, like Bac. sabtilis. Another
illustration of this phenomenon is the growth of the two nitrogen-
fixing organisms, the anaerobic Bac. amylobacter and the aerobic,
rapidly growing Azotobacter. Exposure to oxygen has, however, an
injurious effect upon anaerobic organisms, vegetative cells being de-
stroyed in 10 minutes and spores in 8 days; 14 in the case of Bac. amylo-
bacter; the injurious effect of air exposure upon the vegetative cells
sets in only after 40 minutes, while the spores are not injured even after
3 hour exposure. ^ 5

Methods of isolation of anaerobic bacteria from the soil. There are a
number of methods available for the isolation of anaerobic bacteria. 16
These bacteria have to be separated not only from aerobic organisms,
but often also from other facultative or obligate anaerobic bacteria.
The anaerobes, just as the aerobic bacteria, vary greatly in their food
requirements and manner of growth, and the methods of isolation have
to be adapted to the particular organism in question. There is a large
number of species of anaerobes in the soil and it is insufficient to depend
on microscopic examinations alone for demonstrating the existence of
specific forms. In all cases, the isolation and demonstration of the
different species must be undertaken. In case the nature of an organ-
ism that is looked for is known, the development of a proper culture

13 Hall, I. C. Chemical criteria of anaerobiosis with special reference to
methylene blue. Jour. Bact. 6: 1-42. 1921. Kadisch, E. Centrbl. Bakt. I,
Orig. 90:462-468. 1923. Clark, W. M., Cohen, B., and Gibbs, H. D. Studies on
oxidation-reduction. VIII. Methylene blue. U. S. Publ. Health Serv. Publ.
Health Repts. Repr. no. 1017. 1925.

14 Bachmann, F. Beitrag zur Kenntnis obligat anaerober Bakterien. Cen-
trbl. Bakt. II, 36: 1-41. 1912.

15 Dorner, 1924 (p. 165).

16 Heller, H. H. Principles concerning the isolation of anaerobes. Jour. Bact.
6: 445-470. 1921.

ANAEROBIC BACTERIA 1G5

medium is simplified. An enriched culture is first prepared either by
adding some soil to a specific medium kept under specific conditions,
or the specific substance is added to the soil itself. An attempt is
then made to obtain a culture of the specific bacterium free from accom-
panying non-spore-forming and spore-forming aerobic and anaerobic
organisms.

For the separation of spore-forming organisms from non-spore-
formers, whether aerobes or anaerobes, the soil is heated at 75° to 80°C,
by placing 2 grams of soil in 10 cc. of sterile water and keeping in a water
bath for 10 minutes. This leads to the destruction of all the vegetative
cells, while bacterial spores are not injured. The soil is then inoculated
into a proper medium, favorable for the development of the specific
organism, which will develop under proper cultural conditions; the cul-
ture is then transferred repeatedly upon the selective medium and grown
under strict anaerobic conditions. To purify anaerobes from aerobes,
the method of Dorner 17 can be used. The deep agar tube, inoculated
with the organisms, is allowed to cool and the agar to solidify. Two
cubic centimeters of melted agar containing 0.2 per cent of mercury
bichloride is then poured on the surface of the cooled agar and the
tubes are closed with rubber stoppers. The aerobes are thus completely
eliminated. However, neither of these methods will separate the facul-
tative anaerobes from the obligate anaerobes.

To separate anaerobes from spore-forming aerobes, use is made of
three procedures: (1) Strict anaerobic methods of cultivation. (2)
The inhibitive action of gentian- violet on aerobic growth; 18,19 a 1 : 100,000
to 1:400,000 dilution of the dye in the agar medium is sufficient to
render cultures of anaerobic bacteria free from spore-forming aerobes.
(3) Anaerobic organisms are less sensitive than aerobes to pyrocate-
chin, chinon, sodium formate, and sodium sulphindigotate. 20,21

The most difficult process, often involving a complicated technic, is

17 Dorner, W. Beobachtungen liber das Verhalten der Sporen und vegetativen
Formen von Bac. amylobacter A. M. et Bredemann bei Nachweis- und Reinzucht-
versuchen. Landw. Jahrb. Schweiz. 1924, 1-28.

18 Churchman. The selective bactericidal action of gentian violet. Jour.
Exper. Med. 16: 2, 221, 1912.

19 Hall, I. C. Practical methods in the purification of obligate anaerobes.
Jour. Inf. Dis. 27: 576-590. 1920.

20 Kitasato, S., and Weyl, Th. Zur Kenntniss der Anaeroben. Zeitschr. Hyg.
8: 41, 404. 1890.

21 Rivas, D. Ein Beitrag zur Anaerobenzllchtung. Centrbl. Bakt. 32: 831-
842. 1902.

166 PRINCIPLES OF SOIL MICROBIOLOGY

the separation of spore-forming anaerobes from other spore-forming
anaerobes. The improper separation has led to exaggerated claims for
the nature and activities of the organisms. All or some of the following
procedures are utilized for this separation:

1. Heating the soil so as to kill the vegetative forms, then introducing
various diutions of the heated soil suspension into the proper medium
and making transfers from the culture at different stages of develop-
ment (heating the culture every time a new transfer is made). The
various spore-forming anaerobes sporulate at different periods of their
development: some, like the saccharolytic bacteria, sporulate early;
others, like most proteolytic forms, sporulating late.

2. Use of selective media stimulating the predominant development
of the organism sought. This method has been of great help in the
isolation of some important soil anaerobes. It is sufficient to mention
that by the use of selective media and proper environmental conditions,
such organisms as the anaerobic nitrogen-fixing forms, thermophilic and
cellulose-decomposing forms and others were isolated. The specific
medium is inoculated with an infusion of soil or manure, which may
be previously heated, if the organism in question forms spores, and
incubated at the desired temperature. The adjustment of the medium
to specific reactions may often be sufficient to separate one group of
organisms from another, often even anaerobic forms from one another.
For instance, the adjustment of the nitrogen-free glucose media to a
pH of 5.5 will not only favor the development of the nitrogen-fixing
Clostridium pastorianum, but will also prevent the development of the
proteolytic organisms, which usually accompany it. 22 For the enrich-
ment of cellulose decomposing anaerobic organisms, the use of a specific
liquid medium or of a silica gel plate with cellulose as the only source of
energy is recommended (p. 196). For the decomposisition of hemicel-
lulose, physiological salt solution containing cubes of potato has been
used, 23 while, for starch splitting organisms, media containing 1 per
cent peptone broth and 5 per cent starch have been suggested. 24

3. The use of aniline dyes for the elimination of certain species of
organisms.

4. Selective temperatures for the enrichment of various organisms,

22 Dorner, 1924 (p. 165).

23 Ankersmit, P. Untersuchungen iiber die Bakterien im Verdauungskanal
des Rindes. Centrbl. Bakt. I, Orig, 39: 359-574, 687. 1905; 40: 100-118.

24 Choukevitch, J. Etude de la flore bacte>ienne du gros intestin du cheval.
Ann. Inst. Past. 25: 247. 1911.

ANAEROBIC BACTERIA 167

developing preferably at the different temperatures, as in the case of
thermophilic bacteria.

5. Use of high dilutions for the separation of organisms before
plating. 25

6. Isolation of the individual colony. This can be accomplished
either (a) by the picking of surface colonies from agar or gelatin plates
or slants in large tubes, kept under anaerobic conditions; (6) by picking
colonies from deep agar tubes, 26 the last procedure being the easiest and
most reliable in the process of separation of pure cultures of anaerobic
bacteria from all accompanying forms.

7. Finally the isolation of single cells either by the India ink method, 27
by the method of Barber, or by one of the microscopic methods. 28

A detailed study of the various methods used for the isolation from
surface colonies is given elsewhere. 29,30

In general, plates or large agar slants containing the proper culture
media are streaked out and placed either in vacuo, in hydrogen, carbon
dioxide, or in an atmosphere from which the oxygen is removed by means
of sodium pyrogallate. 31 To produce discreet colonies, the agar plates
or slants must be dried before inoculating, but too much drying of the
medium is injurious. The slants or plates are streaked out with a loop-
ful of the material taken from the enriched culture or using a dilution
of it. The plates are immediately placed in the atmosphere of the neu-
tral gas; the agar may also be placed into the upper part of a Petri dish,
which is then covered directly with the sterile inverted lower half of
the dish and the whole covered with a larger Petri dish. 32

26 Stoddard, J. L. Points in the technic of separating anaerobes. Jour. Am.
Med. Assn. 79: 906. 1918.

26 Burri, R. Zur Isolierung der Anaeroben. Centrbl. Bakt. II, 8: 533-537.
1902.

27 Burri, 1909 (p. 55); also in Krause-TJhlenhut's Handbuch der mikrobiolo-
gischer Technik. 2: 329. 1923.

28 Barber, 1911-1920 (p. 56). Kendall, A. I., Cook, M., and Ryan, M. Methods
of isolation and cultivation of anaerobic bacteria. Jour. Inf. Dis. 29: 227-234.
1921. Holker, J. Micro- and Macro-methods of cultivating anaerobic organ-
isms. Jour. Path. Bact. 22: 28. 1919; 23: 192-195. 1920.

29 von Hibler, E. Untersuchungen liber die pathogenen Anaeroben. Jena.
1908.

30 Besson, A. Practical bacteriology, microbiology and serum therapy. Lon-
don, 1913.

31 Lentz, O. In Friedberger und Pfeiffer's Lehrbuch der Mikrobiologie. Jena,
1919, p. 370.

32 Marino, F. Mcthode pour isoler les anaerobes. Ann. Inst. Past. 21:
1005. 1907; also Ogata, M., and Takenouchi, M. Einfache Plattenkultur-
methode der anaeroben Bakterien. Centrbl. Bakt. I, 73: 75-77. 1914.

168

PRINCIPLES OF SOIL MICROBIOLOGY

fill

However, the deep colony procedure, used first by Liborius for the
isolation of anaerobic bacteria, has been preferred by a number of
workers. The selection of a suitable medium for this purpose is essen-
tial; the medium should be clear and transparent and enough dilution
tubes should be used. Some actively growing anaerobes will grow
through the agar as if it was a broth; this "permeat-
ing growth" will contaminate the other colonies.
The deep tubes of sterile agar are placed in boiling
water till the agar is melted, tubes are shaken to remove
air, and agar cooled down to 45°. Long boiling is
inadvisable, since the cotton becomes saturated with
moisture. Three tubes are employed for ordinary pur-
poses of dilution, but for new material or for weakly
growing organisms among rapidly growing forms, more
tubes may be used. Tube 1 is inoculated with one
loopful of the enriched culture or soil suspension. The
tube is then shaken, and transfer is made by means of a
sterile pipette (a Pasteur pipette may be used), pre-
viously flamed, into tube 2. The inoculum is placed
throughout the length of the agar, while withdrawing
the pipette, taking care not to blow air into the agar in
the tube, the latter being then shaken. The pipette is
flamed and, by means of it, some of the agar from tube
2 is transferred to tube 3, which is also shaken. The
tubes are plugged with cotton, as ordinary aerobic
tubes, and incubated aerobically at 25° to 28°. For
actively growing species, 12 to 24 hours' incubation are
sufficient; for slow growing forms, such as Bac. amylo-
bacter, 4 to 8 days may be required. The
colonies are examined, by means of a hand
lens, for permeating growth and aerobic
organisms. Final isolation is made from
the colonies of the mixed culture. The
tube and colonies to be transferred are
selected. A plain glass or metal rod, steri-
lized in the flame and cooled, may be used
to pierce the agar to the bottom of the tube, so as to admit air and allow
the expulsion of the unbroken agar from the tube upon a sterile half of a
Petri dish. The agar tube may also be placed for a second or two into
warm water so as to separate the agar from the walls of the tube. The

Fig. 9. Buchner tube for
the anaerobic cultivation of
bacteria: p, the alkaline
pyrogallol solution; inner
tube contains culture of or-
ganism (after Omeliansky).

ANAEROBIC BACTERIA 169

agar cylinder is then cut up into fine slices by means of a sterile scalpel;
the desired colony is selected, either with the naked eye or using the
microscope, and the agar is carefully cut away from it. A transfer is
then made by pricking the colony with a fine sterile platinum needle
and inoculating deep tubes with sterile agar or slants and liquid media,
which are then incubated in an oxygen-free atmosphere.

When single cells are separated from one another to obtain pure cul-
tures, it is better to isolate the spores rather than vegetative cells, since
these give a much larger number of successful cultures (Barber). A
medium somewhat more acid than the optimum (as pH 6.0) is more
favorable for the germination of the spores. Semi fluid media (con-
taining 0.1 to 0.2 per cent agar) are preferable to liquid media, since the
presence of a colloid greatly hastens the germination of the bacterial
spores. 33

Further information on the isolation of anaerobic bacteria is given
elsewhere. 34-39

Cultivation of anaerobes. There are a number of methods available
for the cultivation of anaerobes, these methods being largely concerned
with the reduction of the oxygen tension; some of these have been re-
ferred to already previously.

I. Cultivation in the absence of oxygen:

1. Mechanical protection against the atmospheric oxygen. The use of large
volumes of freshly-boiled liquid media placed at a high level; also the process of
covering the media with a laj-er of liquid petrolatum or other inert oil has been
known since Pasteur. A layer of solid medium can be placed in a Petri dish, then
inoculated with anaerobic bacteria and covered with a solution of agar (1.2 to
1.5 per cent) in distilled water. This layer of agar, in covering the medium,
prevents sufficiently the admission of oxygen. The solid medium may also be

33 Lantzsch, 1921 (p. 620).

34 Kursteiner, 1907 (p. 162).

36 Veillon, A., and Maz6, P. De l'emploi des nitrates pour la culture et l'isole-
ment des microbes anaerobies. Compt. Rend. Soc. Biol., 68: 112. 1910.

36 Northrup, Z. A simple apparatus for isolating anaerobes. Jour. Bact. 1:
90-91. 1916.

37 Hort, E. C. The cultivation of anaerobic bacteria from single cells. Jour.
Hyg. 18: 361. 1920.

38 Fuhrmann, F., and Pribram, E. Die wichtigsten Methoden beim Arbeiten
mit Bakterien. Abderhalden's Handb. biol. Arb. Methoden. XII: 483-702.
1924.

39 Lowi, E. Zur Technik der Anaerobenkultur mittels des Pyrogallolverfah-
rens. Centrbl. Bakt. I, Orig., 82: 493-496. 1919.

170 PRINCIPLES OF SOIL MICROBIOLOGY

placed in the upper part of a Petri dish, then covered with the lower part, placed
into the upper part. Solid medium may also be placed in deep layers in ordinary
containers, then inoculated with a long platinum loop reaching to the bottom
of the container (Liborius). The agar can be taken out from the deep tube, by
stabbing to the bottom a sterile glass or metallic tube, 2 mm. in diameter, so as to
admit air. 40

2. Cultivation of anaerobes in vacuo. This method was also proposed by
Pasteur and consists in placing the medium in a tube with a capillary end, inocu-
lating, pumping out the air, then sealing the end. Petri dishes can also be placed
in an ordinary desiccator, from which the atmosphere is then pumped out. The
method described by Meyer 41 can be used for the cultivation of bacteria at differ-
ent partial oxygen tensions.

3. Absorption of oxygen from the atmosphere. The most common method of
absorption of oxygen from the atmosphere is carried out by means of alkaline
pyrogallate solution introduced by Buchner. 42 A mixture of equal portions of
10 per cent solutions of pyrogallol and KOH are often used, or 5 per cent solution
of the first and 12.5 per cent of the second. Buchner used 1 gram of pyrogallic
acid and 10 cc. of 10 per cent solution of KOH for every 100 cc. of air space. 42 The
method of Buchner was modified for liquid media : 44 the sterile cotton plug is pushed
into the tube; 1 cc. of 20 per cent pyrogallic acid and 1 cc. of 20 per cent KOH are
placed upon it, the tube is then closed with a rubber stopper. An alkaline pyro-
catechin FeS04 solution to be used as a sensitive reagent for determining traces
of oxygen has been suggested. 45 The following method is very convenient: 45
About 15 to 20 cc. of agar medium is placed in a large tube, about 1 inch in
diameter, the tube is plugged with cotton and sterilized, then slanted. Imme-
diately after inoculation, the cotton plug is pressed deeply into the tube, about
1 to 2 inches above the tip of slant. One cubic centimeter of a 20 per cent solu-
tion of pyrogallic acid (or a tabloid containing 0.13 gram of the acid) and 0.25 cc.
of a 40 per cent solution of KOH are poured upon the plug, the tube closed
with a rubber stopper, turned upside down and placed in the incubator.

In the case of Petri dishes, Omeliansky used a combination of evacuation and
absorption of oxygen. 43 Ten per cent solution of KOH is poured upon the bottom
of a desiccator and an open Petri dish containing dry pyrogallol is placed in it.
The dishes containing fresh medium and inoculated are then placed into the

40 Burri, R., Staub, W., and Hohl, J. Si'issgriinf utter und Buttersaure-
bazillen. Schweiz. Milchztg. 45: nos. 78, 83. 1919.
"Meyer, 1905 (p. 162).

42 Buchner, H. Eine neue Methode zur Kultur anaerober Mikroorganismen.
Centrbl. Bakt. 4: 149. 1888.

43 Omeliansky, W. L. Ein einfacher Apparat zur Kultur von Anaeroben im
Reagenzglase. Centrbl. Bakt. II, 8: 711-714. 1902.

44 Wright, J. H. A method for the cultivation of anaerobic bacteria. Centrbl.
Bakt. I, 29: 61. 1901.

46 Binder, K., and Weinland, R. F. liber eine neue scharfe Reaktion auf ele-
mentaren Sauerstoff. Ber. deut. chem. Gesell. 46: 255-259. 1913.

46 Buchanan, R. M. An inset absorption appliance for the test-tube culture of
Anaerobes. Centrbl. Bakt. I, Orig., 74: 526-527. 1914.

ANAEROBIC BACTERIA 171

desiccator. The latter is covered and evacuated. The desiccator is then care-
fully turned so as to mix the alkali with the pyrogallol. Since this takes place
in the presence of traces of oxygen it browns only slightly. If the cover is not
tight, the admission of oxygen is readily indicated by the rapid browning of the
mixture. A beaker with water may be placed in the desiccator to prevent the
rapid drying out of the media. 47

Various other methods for the physical or chemical absorption of the oxygen
from the atmosphere have been used; they are based upon the addition of organic
or inorganic substances, possessing a strong reducing power, to the medium or
outside of the medium in a gas-tight vessel. These include ferrous sulfate, sodium
sulfide, ammonium sulf-hydrate, sodium sulfite, ferro-ammonium sulfate, phos-
phorus; glucose, sodium formate, pyrocatechin, indigo-carmin; metallic iron and
zinc; various tissues, pieces of potato, carrot, fresh yeast, etc. These treat-
ments are often accompanied by a partial vacuum. The plates or tubes may be
placed in a container to which a quantity of freshly cut potatoes is added, then
covered with a bell jar.

4. Replacement of air by an indifferent gas. Hydrogen, carbon dioxide, nitro-
gen, and other inert gases may be used for this purpose. A tube, 48,49 flask or
desiccator 50,51 supplied with a two-holed rubber stopper can be used for this
purpose. When all the air is replaced by the inert gas, the tubes are sealed. 62

II. Cultivation in the presence of oxygen:

5. Cultivation of anaerobes in the presence of aerobic organisms. This method
approaches nearest to what takes place in nature than any of the other methods.
By cultivating an anaerobic spore forming organism with an aerobic non-spore
former, like Bad. -prodigiosum, it is easy to obtain a pure culture of the former by
pasteurization. This method has only a limited application in the study of pure
cultures. Beijerinck 53 employed obligate aerobic bacteria to eliminate the last
traces of oxygen from the atmosphere. A combination of two of the above
processes may be used.

The media used for the isolation and cultivation of anaerobic bacteria depend

47 Rockwell, G. E. An improved method for anaerobic cultures. Jour. Inf.
Dis. 35: 581-486. 1924.

48 Fraendel, C. tlber die Kultur anaerober Mikroorganismen. Centrbl.
Bakt. 3: 735, 763. 1888.

49 Petri, R. J., and Maaszen, A. Ein bequemes Verfahren fur die anaerobe
Zuchtung in Flussigkeiten. Arb. K. Ges. Amt. 8: 314. 1893.

60 Botkin, S. Eine einfache Methode zur Isolierung anaerober Bakterien.
Ztschr. Hyg. 9: 383. 1890.

81 Novy, F. G. Die Plattenkultur anaerober Bakterien. Centrbl. Bakt. 16:
566. 1894.

62 Richardson, A. C, and Dozler, C. C. A safe method for securing anaero-
biosis with hydrogen. Jour. Inf. Dis. 31: 617-621. 1922.

63 Beijerinck, M. W. Oidium lactis, the milk mould, and a simple method to
obtain pure cultures of anaerobes by means of it. Proc. Sec. Sci. K. Akad.
Wettensch. Amsterdam, 21: 1219-1226. 1919.

172 PRINCIPLES OF SOIL MICROBIOLOGY

upon the specific organisms. Certain special methods may also be used. Among
these, gelatin and milk have played an important part. By inoculating milk
with a small quantity of soil, a certain type of butyric acid bacteria can be readily
demonstrated. This is not a medium for enrichment of anaerobes, as for differ-
ential purposes. Various protein (egg-albumin) and glucose media can be used.
The medical bacteriologists have made extensive use of brain and blood agar
media. To demonstrate the presence of certain organisms, specific media may
have to be used. To demonstrate the presence of Bac. amylobacter, nitrogen-free
g'.ucose (2 per cent) agar placed in a deep tube is inoculated with a soil sus-
pension; if quantitative results are wanted, various dilutions are employed (the
soil suspension may be previously heated, in a water bath, at 80° for 10 minutes,
whereby only the number of spores is obtained). The tubes are closed with rubber
stoppers (when the culture is to be isolated, a surface layer of sublimate agar is
used) and incubated at 30°. Gas formation will take place on the second day,
breaking up the medium. This and the production of butyric acid will indicate
the presence of the organism ; the colonies are lens-shaped. Microscopic examina-
tion of the culture can be made by staining with Lugol's reagent. The method of
Burri can be used for determining the number of anaerobic bacteria in the soil,
not only by establishing the presence of growth in the final dilution, but by
actually counting the colonies in the deep tube.

Classification of soil anaerobes. Various systems for the classification
of anaerobic bacteria and their relation to aerobes have been proposed
at different times. 54,55 But even at the present time, a proper classifica-
tion of anaerobes, especially of soil forms is lacking. The idea that
anaeobic bacteria vary greatly has served further to increase the exist-
ing confusion. This led to various exaggerations, such as the existence
of only a few anaerobic forms which change into one another, or the
making of new genera on the basis of minor physiological differences. 56

The following system of classification of soil anaerobes may be sug-
gested here merely as a tentative working basis :

I. Bacteria acting primarily upon carbohydrates:

1. Bacteria utilizing largely simple carbohydrates and starches as sources
of energy, often referred to as saccharolytic. Here belong the buty-
ric acid bacteria, often classified as one species, Bac. amylobacter
A. M. et Bred. These decompose sugars with the formation of
butyric acid and gas :
(a) Nitrogen-fixing bacteria — ■Clostridium pastorianum Winogradsky
(Bac. amylobacter von Tieghem, Bac. amylocyme Perdrix,
Bac. butyricus Botkin, Granulobacler saccarobutyricum Beij.,
Bac. orthobutylicus Grimbert, Clostridium americanum Pring-

" Hibler, 1908 (p. 167); Bredemann, 1909 (p. 109).
65 Jungano, M., and Distaso, A. Les anaerobies. Paris. 1910.
"Heller, H. H. Classification of the anaerobic bacteria. Bot. Gaz. 73:
70-79. 1922; Jour. Bact. 7: 1-38. 1922.

ANAEROBIC BACTERIA 173

sheim, Bac. amylobacter A. M. Bred.) and allied forms found
in great abundance in practically all soils. This organism
or group was classified by Bergey as CI. butyricum Prazmowski
and by Lehman and Neumann as Bac. pastorianus (Winograd-
sky). 57 Its physiology and occurrence in the soil is discussed
elsewhere (p. 110).
(b) Bac. welchii Migula (Bac. aerogenes capsulalus Welch and Nutall,
Bac. perfringens Veillon and Zuber, Bac. enteritidis sporogcnes
Klein), a short rod 4 to 8 by 1 to 1.5/u, single or in pairs; non-
motile, forming oval, central or excentric spores; encap-
sulated (No. 44, PI. IX). Found repeatedly in the soil and
in sewage. 58

2. Bacteria decomposing pectins.

(a) Bac. amylobacter group, which includes the Clostridium pastor i-

anum (same as la). The forms causing the retting of flax
have been described under various names. Here belong the
Plectridium of Fribes and Winogradsky, the Clostridium of
Behrens, the Plectridium pectinovorum of Stormer, the
Granulobacter pectinovorum of Beijerinck and van Delden. 19

(b) Bac. felsineus Carbone.

3. Bacteria decomposing celluloses:

(a) Anaerobic bacteria decomposing celluloses at ordinary temper-

atures. Here belong the hydrogen and methane organisms
of Omeliansky and the Bac. cellulosae dissolvens Khouvine.

(b) Thermophilic cellulose decomposing bacteria — Clostridium ther-

mocellum Viljoen, Fred and Peterson. The occurrence and
isolation of these organisms is described elsewhere (p. 202).
II. Bacteria acting primarily upon proteins:
1. Strongl y proteolytic forms:

(a) Bac. sporogenes Metchnikoff (No. 58, Pi. X), a motile, flagellated,
gram positive bacillus, with rounded ends, 3 to 7 by 0.6 to
0.8m,* one of the strongest proteolytic bacteria known; it de-
composes proteins with the formation of gas, a darkening
of the medium and production of a pronounced odor; the sub-
terminal spores are formed readily. Found abundantly in the
soil, manure, street dust and sewage.

67 Further information on the classification of the anaerobic bacteria acting
primarily upon carbohydrates is given by Donker, H. J L. Bijdrage tot de
kennis der Baterzuur — , Butylalcohol — en Acetongistingen. Delft. 1926.

68 Klein and Houston. Rept. Med. Officer, Local Govt. Board, London, 1898-
1899, 318; Greer, F. E. Anaerobes in sewage. Amer. J. Publ. Health, 15: 860-867.
1925.

69 Ruschmann, G., and Ravendamm, W. Zur Kenntnis der Rosterreger Bacil-
lus fehineus Carbone und Plectridium pectinovorum [Bac. amylobacter A. M. et
Bredemann). Centrbl. Bakt. II, 64: 340-394. 1925.

174 PRINCIPLES OF SOIL MICROBIOLOGY

(b) Bac. oedematis maligni Koch (Vibrion septique Pasteur), found

in the intestines of man and in the soil. 60

(c) Bac. putrificus Bienstock, a motile, flagellated bacillus, forms

terminal oval spores and has weak saccharolytic and strong
proteolytic properties. Milk is gradually digested, without
rapid coagulation (No. 45, Pi. IX).

(d) Bac. histolyticus Weinberg and Seguin, 3.0 to 5.0 by 0.5 to 0.7/u,

occurring singly or in pairs, motile by peritrichous flagella;
spores oval excentric. This organism has been isolated from
the soil by Peterson and Hall. 61

(e) Bac. botulinus van Ermengem, large rods with rounded ends;

oval, subterminal spores. The natural habitat of this
organism has been found in virgin and cultivated soils,
mountain and forest soils, 62-64 throughout the world.
2. Weakly proteolytic organisms:

(a) Bac. bijermentans Tissier and Martelly, non-motile bacillus, with

large central, oblong to oval spores.

(b) Bac. telani Nicolaier; 4 to 8 by 0.4 to 0.6/x," motile by means of

peritrichic flagella; unable to utilize carbohydrates, intro-
duced into the soil with the manure. 65 Its occurrence in the
soil has been demonstrated 64,66 in many of the samples
examined.

Various other anaerobic bacteria which are weakly pro-
teolytic, but are capable of attacking different carbohydrates,
with the formation of gas have been isolated either directly
from the soil or from other sources, which may indicate a soil
habitat, such as Bac. chauvoei. A detailed study of the
various anaerobic bacteria, including Bac. sporogenes, Bac.
histolyticus and others, secured from wound infections and

60 Gt. Britain National Health Ins. Joint Comm., Medical Research Com-
mittee. Special Reports, Series No. 39. Reports of the Committee upon anae-
robic bacteria and infections. 1919.

61 Peterson, E. C, and Hall, I. C. The isolation of Bacillus histolyticus from
soil in California. Proc. Soc. Exp. Biol. Med. 20: 502-503. 1923.

62 Tanner, F. W., and Dack, G. M. Clostridium botulinum. Jour. Inf. Dis.
31: 92-100. 1922.

63 Dubovsky, B. J., and Meyer, K. F. An experimental study of the methods
available for the enrichment, demonstration of B. botulinus in specimens of soil,
etc. Jour. Inf. Dis. 31: 501-540, 541-555, 556-558, 559-594, 595-599, 600-609,
610-613. 1922.

64 Hall, I. C, and Peterson, E. C. The detection of Bacillus botulinus and
Bacillus tetani in soil samples by the constricted tube method. Jour. Bact. 9:
201-209. 1924.

66 Noble, W. Experimental study of the distribution and habitat of the tet-
anus bacillus. Jour. Inf. Dis. 16: 132-141. 1915.

66 Dubovsky, S. J., and Meyer, K. F. The occurrence of B. tetani in soil
and on vegetables. Jour. Inf. Dis. 31: 614-616. 1922.

ANAEROBIC BACTERIA 175

probably coming in most cases originally from the soil has
been made by Weinberg and Seguin. 67
III. Bacteria obtaining their oxygen from inorganic salts:

1. Bacteria reducing nitrates.

2. Bacteria reducing sulfates.

Both of these groups are described in detail elsewhere (p. 180).

Anaerobic organisms may occur in the soil in great abundance;
Ucke 68 found a garden soil to contain 13| million cells of anaerobic bac-
teria and 500,000 spores per 1 gram of soil. In some cases individual
species are found in the soil in great abundance. Kiirsteiner, for exam-
ple, found as many as 1 million and more cells of Bac. putrificus per 1
gram of soil. Bac. amylobacter was found by Bredemann to be present
in practically every soil examined, both in the surface layer and in the
subsoil, in cultivated soils, in primeval forests and in pure sand; the
organism occurred only irregularly in acid peat soil. Out of 200 sam-
ples of Swiss soils examined, only seven did not contain this organism. 69
The number of colonies formed on artificial media are considerably less
than the actual number of organisms actually present in the soil; this is
brought out by the results of Dorner, 69 who found that out of 1000
spores present in a medium, only 3 germinated and developed into
colonies, while out of 1000 vegetative cells, 45.1 produced colonies.

By the use of the dilution and selective culture method, Duggeli 70
found 1000 to 1,000,000 anaerobic butyric acid bacteria per gram of
soil, to 1000 anaerobic cellulose-decomposing bacteria, 100 to 1,000,000
anaerobic nitrogen-fixing bacteria, from 100 to 1,000,000 anaerobic
protein-decomposing bacteria and 100 to 1,000,000 pectin-decomposing
bacteria. By the deep tube method, only between 19,000 and 900,000
anaerobic bacteria were found per gram of soil. This is due to the
fact that no single solid medium can be devised which would be favorable
for the development of all anaerobic bacteria.

Various anaerobic bacteria take an active part in the composting
of manure in the heap, whenever there is an insufficiency of aeration.
The so-called phenomenon of "putrefaction" is chiefly a result of the
decomposition of protein substances under anaerobic conditions, due to

67 Weinberg, M., and Seguin, P. La gangrene gazeuse. Masson & Cie. Paris.
1918.

68 Ucke, A. Ein Beitrag zur Kenntnis der Anaeroben. Centrbl. Bakt. I, 23:
996-1001. 1898.

"Dorner, 1924 (p. 165).
70 Duggeli, 1921 (p. 39).

176 PRINCIPLES OF SOIL MICROBIOLOGY

incomplete oxidation as a result of insufficient aeration. The absence
of air in the deeper piles of manure, the slightly alkaline reaction and the
presence of large amounts of undecomposed substances make conditions
favorable for the development of anaerobic bacteria. 71 Various anaero-
bic urea bacteria 7 ^ and thermophilic organisms 73 also find conditions in
the composting manure heap favorable for their development. Well
rotted horse manure contains spore-forming, anaerobic thermophilic bac-
teria; 74 the limiting temperature for their growth was found to be 60° to
65°C. and the thermal death point 110° to 120°C. Some of these or-
ganisms were found to be actively proteolytic. No growth took place
at room temperature. Various anaerobic spore-bearing bacteria are
no doubt brought into the soil with the feces in great abundance; a
number of these organisms have actually been demonstrated in intes-
tinal secreta. 75

Physiological activities of anaerobic bacteria. It is impossible to dis-
cuss the physiological activities of the various obligate anaerobic bac-
teria, since they differ greatly in the nature of their metabolism. Those
that obtain their energy from cellulose, those that can obtain their
nitrogen from the elementary form, those that can utilize nitrate or
sulfate oxygen, and those that produce foul odors from complex proteins
have a distinct physiology from one another and cannot be considered
under one heading, merely because they are similar in their requirements
of oxygen tension. They usually have an optimum range of hydrogen-
ion concentration at pH 6.0 to 8.2 with a limiting range of pH 5.0 to
9.0; the spores germinate better at a higher acidity, with an optimum at
pH 6.0 to 7.2. 76

While aerobic bacteria produce largely carbon dioxide among the
volatile gases, the anaerobic bacteria are characterized by the production

71 Severin, S. A. Die im Miste vorkommende Bakterien und deren physiolo-
gische Rolle bei der Zersetzung derselben. Centrbl. Bakt. II, 1: 799-817. 1895;
3: 628-633, 708. 1897. Zhur. Opit. Agron. (Russian), 1: 463-489. 1920.

"Geilinger, 1917 (p. 210).

73 Veillon, R. Sur quelques microbes thermophiles strictement anadrobies.
Ann. Inst. Past. 36: 422-438. 1922.

74 Damon, S. R., and Feiber, W. A. Anaerobic sporulating thermophiles.
Jour. Bact. 10: 37-46. 1925.

75 Kahn, M. C. Anaerobic spore-bearing bacteria of the human intestine in
health and in certain diseases. Jour. Inf. Dis. 35: 423-478. 1924.

76 Dozier, C. C. Optimum and limiting hydrogen-ion concentrations for
B. botulinus and quantitative estimation of its growth. Jour. Inf. Dis. 35: 105-
133. 1924.

ANAEROBIC BACTERIA 177

of a number of other gases. It is sufficient to mention hydrogen and
methane, as a result of decomposition of carbohydrates, hydrogen sul-
fide as a result of reduction of sulfates, elementary nitrogen and oxides
of nitrogen as a result of reduction of nitrates, and various amines,
elementary nitrogen and oxides of nitrogen, hydrogen sulfide, mercap-
tans and thioether as a result of decomposition of proteins. It is neces-
sary to be able to measure these and determine them quantitatively,
especially since they are often of great economic importance when a soil
is water-logged for a longer or shorter period of time. The bacteria
are grown on suitable media (specific for the various organisms)
under anaerobic conditions, in tubes or bottles connected with a manom-
eter. The tubes may also be placed in a Novy jar used as a respiratory
chamber. 77 The growth may be carried on in an atmosphere of pure
gas, such as N 2 , H 2 , C0 2 . By using a compensation manometer, the
pressure changes taking place within the culture tube or jar can be
observed constantly, these changes indicating the periods of active
growth followed by the cessation of growth and respiration. The
samples of gas are withdrawn directly into a burette or first into a
sampler, then into a modified Henderson-Haldane or other suitable
apparatus.

The volume of the gas to be analyzed is first measured; the gas is
then passed back and forth into 10 per cent KOH solution to absorb the
C0 2 , which is determined by difference in the volume of gas. The latter,
freed from C0 2 , is passed into an alkaline pyrogallate solution (or
sticks of yellow phosphorus in water) to absorb the oxygen; the latter
is determined also by the difference in volume of the gases. The
estimation of hydrogen, methane and other combustible gases is carried
on in a combustion chamber over heated platinum, in the presence of
oxygen (or air as a source of oxygen). By measuring the amount of
C0 2 formed in combustion, it is possible to calculate the amount of
methane and other hydrocarbons present in the gas mixture; the amount
of hydrogen is then determined by the difference between the loss due
to combustion and the methane present. The amount of oxygen
absorbed in the combustion is obtained by calculation or by the differ-
ence between the oxygen added and that remaining, as determined by
absorption in the pyrogallate solution. The C0 2 present in the medium
(liquid) is aerated into standard Ba(OH) 2 solution, then titrated.

77 Novy, F. G., Roehm, H. R., and Soule, M. H. Microbic respiration. I.
The compensation manometer and other means for the study of microbic respira-
tion. Jour. Inf. Dis. 36: 109-167. 1925.

178 PRINCIPLES OF SOIL MICROBIOLOGY

Oxides of nitrogen are determined by combustion in the platinum
spiral before oxygen (or air) is admitted, in the presence of hydrogen.
The contraction in volume serves as an index of N 2 (N 2 + H 2 — *
H 2 + N 2 ). The oxides of nitrogen may be absorbed from 100 cc.
sample of gas in 200 cc. m/50 KOH solution, then oxidized to nitrate
by adding 5 cc. of 30 per cent hydrogen peroxide. The solution is evapo-
rated to dryness on a water bath and nitrates determined by the phe-
noldisulphonic acid method. 78 Volatile amines and mercaptans do not
occur in great abundance among the decomposition products in the soil,
but are found largely in the anaerobic decomposition of manure: 79,80
H 2 S gas can be determined by absorption with acetates of lead and cad
mium, or ammoniacal cadmium chloride solution, then titrating the
CaS with iodine in acid solutions. 81

Among the gases formed by the proteolytic bacteria, like Bac. sporo-
genes, we find largely C0 2 and some hydrogen; the odoriferous gases
consist largely of H 2 S; some elementary nitrogen and N 2 are also
formed. The saccharolytic organisms, like Bac. welchii, produce a
large amount of hydrogen, often as much as 50 per cent of the gases. 81
The ratio between the C0 2 and hydrogen depends largely upon the
environmental conditions of growth.

Anaerobic bacteria form various acids (acetic, butyric, lactic), alco-
hols (ethyl, butyl), and in some cases acetone. Often closely related or-
ganisms vary greatly in their metabolic products. For example, while
different members of the Bac. amylobacter group (Clostridia, Plectridia,
Granulobacter) produce butyric acid, the closely related Bac. felsineus
does not do so.

Soil processes in which anaerobic bacteria take an active part. Atten-
tion has already been called to a number of important physiological
processes in the soil, in which anaerobic bacteria take an active part.
It is sufficient to indicate that such processes as decomposition of cellu-
loses, pectins and proteins, and the fixation of nitrogen non-symbioti-
cally are as active anaerobically as aerobically. Ammonia formation

78 Allison, V. C, Parker, W. L., and Jones, J. W. Determination of oxides of
nitrogen. Tech. Paper No. 249, U. S. Bureau of Mines. 1921.

79 Guggenheim, M. Die biogenen Amine. 1920.

80 Hirsh, P. Die Einwirkung von Mikroorganismen auf die Eiweisskorper.
Borntraeger. Berlin. 1918.

81 Anderson, B. G. Gaseous metabolism of some anaerobic bacteria. XIX.
Methods. Jour. Inf. Dis. 35: 213-243. 1924.

ANAEROBIC BACTERIA 179

from proteins is very active under anaerobic conditions. 82-84 Two
maxima were found for nitrogen-fixation in the soil, one under aerobic
and another under anaerobic conditions; 83 - 86 a higher fixation may
actually be obtained anaerobically. 87 The decomposition of cellulose
under anaerobic conditions is carried on entirely by bacteria. The
phenomena of reduction under anaerobic conditions, especially that of
nitrates, may become an important economic factor.

It is important to point out, in this connection, the active role
which anaerobic bacteria play in the rotting of manure. As a matter of
fact, the lowest loss of nitrogen and the most efficient conservation of the
important elements of the manure is accomplished by keeping it com-
pact and moist, so as to prevent the action of aerobic fungi and bacteria
and stimulate the action of anaerobic bacteria. As far back as 1889,
Schloesing 88 pointed out that under anaerobic conditions there is no
loss of nitrogen. The gases were found to consist of equal volumes of
methane and carbon dioxide, when the manure is incubated at 52°C.
Water takes part in the reaction supplying some oxygen for the forma-
tion of C0 2 and some hydrogen for the methane. The amount of gas
produced per hour rapidly increases until it reaches a maximum on the
6th day, when it begins to diminish. At 42°C, 850 cc. of gas collected
from the decomposition of 100 gm. of manure consisted of 713.6 cc.
C0 2 , 97.6 cc. methane and 38.8 cc. hydrogen.

Further information on the decomposition of proteins and carbo-
hydrates under anaerobic conditions and on the nature of soil gases is
given elsewhere (p. 638).

» 2 L6hnis, 1905 (p. 120).

83 Traaen, A. E. Uber den Einfluss der Feuchtigkeit auf die Stickstoff um-
setzungen im Erdboden. Centrbl. Bakt. II, 45: 115. 1916.

84 Murray, T. J. The oxygen requirements of biological soil processes. Jour.
Bact. 1: 597-614. 1916.

85 Greaves, J. E. Azofication. Soil Sci. 6:163-218. 1918.

86 Lipman and Sharp, 1915 (p. 584).

87 Panganiban, E. H. Rate of decomposition of organic nitrogen in rice paddy
soils. Phillip. Agriculturist, 12: 63. 1923; Temperature as a factor in nitrogen
changes in the soil. Jour. Amer. Soc. Agron. 17: 1-31. 1925.

88 Schloesing, 1889 (p. 62).

CHAPTER VII
Bacteria Reducing Nitrates and Sulfates

General classification of nitrate reducing bacteria. A large number of
organisms, including numerous bacteria and actinomyces, fungi,
yeasts and higher plants, but especially the first two groups, are capable
of reducing nitrates to nitrites; this often serves as the first step in the
process of assimilation of nitrate nitrogen. Some organisms, chiefly
fungi and certain bacteria, but also higher plants, are capable of reduc-
ing the nitrate to ammonia. However, only specific bacteria are cap-
able, under certain conditions, of reducing the nitrate and the nitrite
to elementary nitrogen and oxides of nitrogen, in which form the nitro-
gen escapes into the atmosphere. Under anaerobic conditions, the
nitrate and nitrite may serve as sources of oxygen for these bacteria,
which enables them to oxidize the available sources of energy. 1 The
last process is usually referred to as complete or direct denitrification and
the bacteria concerned in this process are spoken of as denitrifying
bacteria. These bacteria can be further subdivided into (a) those which
use as a source of energy inorganic substances, notably sulfur, and (6)
those that use organic carbon compounds as sources of energy. Com-
plete denitrification is generally favored by the presence of nitrate,
suitable sources of energy (usually carbon compounds), absence of free
oxygen and proper reaction.

The bacteria, which reduce nitrates only to nitrites or to ammonia,
but not to nitrogen gas (elementary form and oxides), may be best
spoken of as nitrate reducing bacteria, reserving the term denitrifying
bacteria for the other organisms.

Organisms reducing nitrates to nitrites. The reduction of nitrates in
the soil has been demonstrated in the first part of the 19th century.

1 Weissenberg, H. Studien uber Denitrifikation. Arch. Hyg. 30:279-290.
1897; Jensen, H. Das Verhaltnis der denitrifizierenden Bakterien zu einigen
Kohlenstoffverbindungen. Centrbl. Bakt. II, 3: 622-627, 689-698. 1897; Bei-
trage zur Morphologie und Biologie der Denitrifikationsbakterien. Ibid. 4:
401-411, 449-460. 1898; Pakes, W. C. C, and Jollyman, W. H. The collection
and examination of the gases produced by bacteria from certain media. Jour.
Chem. Soc. I, 79: 322-329. 1901.

180

BACTERIA REDUCING NITRATES AND SULFATES 181

This process was found to be brought about by various groups of micro-
organisms, capable of reducing nitrates to nitrites, first by Schonbein 2
in 1868, then by others, especially by Gayon and Dupetit. 3 In addition
to various bacteria, 4 certain yeasts, filamentous fungi, 5 and actinomyces 6
are capable of reducing nitrates to nitrites. The composition of the
medium is important in this respect, particularly the nature of other
sources of nitrogen and of the energy source. The presence of carbo-
hydrates, glycerol and organic acids, in addition to peptone, was found
to stimulate the reduction of nitrate to nitrite, while an abundance of
oxygen injured it.

Frankland 7 called attention to the fact that certain bacteria {Bac.
ramosus and Bac. pestifer) are specifically concerned in this process.
The reduction was favorably influenced by increasing the organic
matter content of the solution, especially the peptone. Anaerobiosis
or lack of sufficient aeration greatly favors nitrite formation. 8-10
Nitrite-forming bacteria are well distributed in the soil. 11,12 Such soil
forms as Bac. megatherium 13 and Bad. vulgare u are found among the

2 Schonbein, C. F. tJber die Umwandlung der Nitrate in Nitrite durch Confer-
ven und andere organische Gebilde. Jour, prakt. Chem. 105: 208-214. 1868.

3 Gayon, U., and Dupetit, G. Sur les fermentations des nitrates. Compt.
Rend. Acad. Sci. 95 : 644-646. 1882; Sur la transformation des nitrates en nitrites.
Ibid., 1365-1367; Recherches sur la reduction des nitrates par les infiniments
petits. Nancy. 1886; Mem. Soc. Sci. phys. Nat. Bordeaux. 1886; Ann. Sci.
Aeron. 1: 226-325. (1885) 1886.

4 Maassen, A. Die Zersetzung der Nitrate und Nitrite durch die Bakterien.
Arb. K. Gesundheitsamt, 18: 21-77. 1901.

5 Wolff, K. Denitrifikation und Garung. Hyg. Rundschau, 91: 538. 1899.

6 Waksman, 1919 (p. 299).

7 Frankland, P. J. The action of some specific microorganisms on nitric acid.
Chem. News, 57: 89. 1888; Uber einige typische Mikroorganismen im Wasser
und im Boden. Ztschr. Hyg. 6: 373. 1899.

8 Laurent, E. Experiences sur la reduction des nitrates par les vegetaux.
Ann. Inst. Past. 4: 722-744. 1890.

9 Kiihl, H. Beitrag zur Kenntnis des Denitrifikationsprozesses. Centrbl.
Bakt. II, 20: 258-261. 1908.

10 Caron, H. V. Untersuchungen uber die Physiologie denitrifizierender
Bakterien. Centrbl. Bakt. II, 33: 62-116. 1912.

11 Jensen, 1897 (p. 180).

12 Klaeser, M. Die Reduktion von Nitraten zu Nitriten und Ammoniak durch
Bakterien. Centrbl. Bakt. II, 41: 365-430. 1914: Ber. deut. bot. Gesell. 32:
58. 1914.

13 Stoklasa, 1898 (p. 104).

14 Horowitz, A. Contribution a l'£tude du geare Proteus vulgaris. Ann. Inst.
Past. 30: 307-318. 1916.

182 PRINCIPLES OF SOIL MICROBIOLOGY

nitrite formers. Out of 109 species of bacteria tested by Maassen, 15
in a solution containing 5 per cent peptone and 0.5 per cent sodium ni-
trate, 85 were found capable of reducing nitrates to nitrites, especially
Bad. pyocyaneum; 46 reduced the nitrite to ammonia and 4 liberated
atmospheric nitrogen. Out of 28 species of bacteria studied by Klae-
ser, 12 all but one were found capable of reducing nitrates. Many
strict aerobic bacteria are capable of acting anaerobically in the pres-
ence of nitrates. Intensive aeration inhibits the process of nitrate re-
duction. The reaction of the medium has an important influence in
determining whether nitrates are reduced to nitrites or ammonia; an
alkaline reaction favors the first process and an acid reaction the
second.

Klaeser used a medium having the following composition :

KN0 8 2 grams NaCl 0.1 gram

Glucose 10 grams MgS0 4 0.3 gram

K 2 HP0 4 1 gram FeCl 3 0.01 gram

CaCl 2 0.1 gram

Other media, with and without peptone, but containing nitrates, can also be
used for demonstrating nitrate reduction by bacteria. The formation of nitrites
from nitrates has been suggested as a test in characterizing bacteria. 16

The following organisms can be recorded as capable of reducing
nitrates to nitrites: Bad. coli, Bad. vulgare and allied species, Bad.
prodigiosum, Bad. putidum, Bad. fluorescens, Bad. pyocyaneum, Bad.
herbicola, Bac. subtilis and allied species, Bac. vulgatus, Bac. mycoides,
Micr. pyogenes, Mycobad. phlei and other mycobacteria, B. porticensis
and others. Some of these organisms, such as Bad. coli, are also cap-
able of forming hydrogen. 17 The products formed from the reduction of
the nitrate depend largely upon the composition of the medium and
oxygen tension.

Organisms reducing nitrates to ammonia. Marchal 18 was one of the
first to demonstrate that certain bacteria (Bac. mycoides) are capable of
reducing nitrates to ammonia, with the intermediate formation of ni-

15 Maassen, 1901 (p. 181).

16 Conn, H. J., and Breed, R. S. The use of the nitrate-reduction test in char-
acterizing bacteria. Jour. Bact. 4: 267-290. 1919.

17 Maze, P. Les phdnomenes de fermentation sont les actes de digestion nou-
velle demonstration apport£e par l'etude de la devitrification dans le regne vegetal.
Ann. Inst. Past. 25: 289-312, 369-391. 1911.

18 Marchal, E. The production of ammonia in the soil by microbes. Agr.
Sci. 8: 574. 1S94; Centrbl. Bakt. II, 1: 758. 1895.

BACTERIA REDUCING NITRATES AND SULFATES 183

trites. Beijerinck and van Delden 19 found that various bacteria, like
Bac. subtilis and Bac. mesentericus vulgatus, are capable of producing
both ammonia and nitrite from nitrates, but no ammonia from nitrites;
Azotobacter chroococcum, however, produced ammonia from nitrates and
nitrites. The reduction process takes place in the presence of carbo-
hydrates and organic acids as sources of carbon. 20 These bacteria
undoubtedly include the "protein-forming bacteria" described by
Gerlach and Vogel, 21 capable of transforming nitrate into protein nitro-
gen with an intermediate reduction to ammonia nitrogen.

Kruse 22 called attention to the fact that those microorganisms, which
cannot bring about "fermentation of the nitrate" (complete reduction
to nitrogen), are capable of reducing it to ammonia. This seems to be
the natural process, when microorganisms are assimilating nitrates and
nitrites, to reduce them first to ammonia, as shown for a number of
bacteria and fungi. 23

Bacteria reducing nitrates to atmospheric nitrogen. The formation of
gaseous nitrogen in the process of decomposition of organic matter in
the soil was first observed by Davy. 24 This was ascribed to a chemical
interaction between nitrites and amino acids in the soil, resulting in
the formation of gaseous nitrogen. 25 Gayon and Dupetit 26 pointed out
in 1882 that bacteria were responsible for this process and that the free
nitrogen originated from the nitrates. Deherain and Maquenne 27
demonstrated that nitrate decomposition in the soil takes place only in
the absence of atmospheric oxygen and in the presence of an abundance

19 Beijerinck, M. W., and van Delden, A. tJber die Assimilation des freien
Stickstoffs durch Bakterien. Centrbl. Bakt. II, 9: 3-43. 1902.

20 Stoklasa, J., and Vitek, E. Beitrage zur Erkenntnis des Einflusses verschie-
dener Kohlenhydrate und organischer Sauren auf die Metamorphose des Nitrats
durch Bakterien. Centrbl. Bakt. II, 14: 102-118. 1905.

21 Gerlach and Vogel. tlber eiweissbildende Bakterien. Centrbl. Bakt. II,
7: 609-623. 1901.

22 Kruse, 1910 (p. xii).

23 Kostyschew, S., and Tswetkowa, E. Uber die Verarbeitung der Nitrate in
organische Stickstoffverbindungen durch Schimmelpilze. Ztschr. physiol.
chem. Ill: 171-200. 1921.

24 Davy, 1814 (p. 122).

26 Dietzell, B. E. Ueber die Entbindung von freien Stickstoff bei der Faulnis.
Ztschr. Landw. Ver. Bayern. 72: 186 201. 1882. (Biederm. Centrbl. Agrik.
Chem. 11: 417-420. 1882).

26 Gayon and Dupetit, 1882 (p. 181).

27 Deh6rain, P. P., and Maquenne. Sur la reduction des nitrates dans la terre
arable. Compt. Rend. Acad. Sci. 95: 691-693, 732-734, 854-856. 1882.

184 PRINCIPLES OF SOIL MICROBIOLOGY

of organic matter. The process is checked by heating the soil or treat-
ing it with chloroform, which results in the destruction of the bacteria
responsible for the reduction of the nitrates. 28 In the decomposition of
organic nitrogenous compounds, free from nitrates, both in the presence
and absence of oxygen, nitrogen gas is not produced; when nitrates are
present, an active reduction takes place in the absence of oxygen, with
the formation of gaseous nitrogen and various oxides of nitrogen. 29
This reduction diminishes with an increase in the amount of oxygen
present but does not stop entirely. Even those investigators who
believed at first that denitrification is a purely chemical process, carried
out by means of the soil colloids, were convinced by later studies that
nitrate reduction is not of a chemical nature. 30

Bacteria may bring about the formation of nitrogen gas from nitrates
in two different ways: (a) indirectly and (b) directly. The nitrite
which is formed in the process of reduction of nitrate by Bad. coli,
Bad. vulgare, Bad. prodigioswn, Bac. vulgatus, may interact chemically
with the amino nitrogen of the peptone molecule or the various amino
acids formed from the decomposition of peptone, liberating gaseous
nitrogen. The various oxides of nitrogen formed from the reduction of
nitrate may also interact with the ammonia nitrogen formed from the
peptone and result in free nitrogen gas:

NH 4 N0 2 = 2H 2 + N 2

These indirect processes play only a questionable role in the soil. How-
ever, in addition to these bacteria, which in themselves are unable to
produce nitrogen gas directly from nitrates, the soil harbors various
specific bacteria capable of reducing the nitrate molecule directly to
atmospheric nitrogen. Breal 31 found that a nitrate solution to which
straw is added liberates a great deal of gaseous nitrogen. Similar
results have been obtained on inoculating a nitrate solution with horse

28 Ehrenberg, A. Experimentaluntersuchungen iiber die Frage nach dem Frei-
werden von gasformigen Stickstoff bei Fiiulnissprocessen. Ztschr. physiol.
Chem. 11: 145-178, 438-471. 1886.

2D Tacke, Br. tJber die Entwicklung von Stickstoff bei Fiiulniss. Landw.
Jahrb. 16: 917-939. 1888.

30 Vogel, J. tiber das Verhalten von Nitrat im Ackerboden. Centrbl. Bakt.
II, 34: 540. 1912; Landw. Vers. Sta. 78: 265-301. 1912; 82: 159-160. 1913.

31 Breal, E. De la presence dans la paille d'un ferment a^robie r<5ducteur de
l'acide nitrique. Ann. Agron. 18: 181-195. 1892. Compt. Rend. Acad. Sci.
114: 681-684. 1892.

BACTERIA REDUCING NITRATES AND SULFATES 185

manure. Wagner 32 then attempted to draw, on insufficient ground,
broad generalizations concerning the reduction of nitrates to gaseous
nitrogen by denitrifying bacteria in manure, even when added to the
soil.

Gayon and Dupetit 33 isolated from the soil, in 1886, two anaerobic
bacteria (B. denitrificans a and jS) capable of reducing nitrates to gaseous
nitrogen. The two organisms were cultivated upon a medium having
the following composition: 34

1. Distilled water 250 cc. 2. Distilled water 500 cc.

KN0 3 2 grams Citric acid 5 grams

Asparagine 1 gram KH 2 PO 4 2 grams

MgS0 4 2 grams

CaCl 2 0.2 gram

FeCl 3 Trace

Solution 2 is neutralized with a 10 per cent solution of NaOH or KOH, with
phenolphthalein as an indicator. The two solutions are mixed and made up to
1000 cc. with distilled water.

For the isolation of denitrifying organisms, various other media can be used:
(1) 1000 cc. water, 10 grams glucose, 6 grams NaN0 3 , 6 grams NaCl, 0.02 gram
Ca 3 (P0 4 ) 2 . 35 (2) 100 cc. water, 0.5 to 1.5 grams NaN0 3 , 20 to 50 grams glycerol,
7 grams malic acid (neutralized with sodium carbonate), 0.5 gram sodium phos-
phate, 0.5 gram NaCl, 0.5 gram Na 2 C0 3 , 0.1 gram MgS0 4 . 36 (3) 1000 cc. water,
20 grams of calcium tartrate, citrate or malate, 10 to 20 grams KN0 3 , 0.5 gram
K 2 HP0 4 .

Under anaerobic conditions, practically all the nitrate nitrogen can
be transformed into gaseous nitrogen. When asparagine is replaced by
sugar, the ammonia otherwise produced from the asparagine is not
formed. In the reduction of nitrate to gaseous nitrogen (so-called "ni-
trate fermentation"), there is an abundant accumulation of alkali, till
the process is stopped when the alkali concentration is equivalent to
1 per cent sodium carbonate. 37 When the alkali is neutralized by means

32 Aeby, J., Dorsch, R., and Matz, Fr., and Wagner, P. Forschungen iiber
den relativen Dungewart und die Konservierung des Stallmistsickstoffs. Landw.
Vers. Sta. 48: 247-360. 1897.

33 Gayon and Dupetit, 1886 (p. 181).

34 Giltay, E., and Aberson, G. Denitrifizierende Organismen im Boden.
Arch. Neerland. 25: 341. 1892.

36 Ampola and Ulpiani. Gazz. chim. ital. 1898, 410.
36 Maassen, 1901 (p. 181).

37 Burri, R., and Stutzer, A. Uber Nitrat zerstorende Bakterien und den durch
dieselben bedingten Stickstoffverlust. Centrbl. Bakt. II, 1: 257-265, 350-364,
392-398, 422-432. 1895; 2: 473-474. 1896.

186 PRINCIPLES OF SOIL MICROBIOLOGY

of an acid, nitrate reduction continues further, until all the nitrate has
disappeared. 38 The organisms are very sensitive to free acids. The
optimum reaction for the reduction of nitrates is pH 7.0 to 8.2; the limit-
ing reactions are pH 5.5 and pH 9.8. The optimum reaction for the
reduction of nitrites is pH 5.5 to 7.0.

The reduction of nitrates to atmospheric nitrogen may be a result of
associative action of two bacteria, one (Bad. coli) reducing the nitrate
to nitrite and the other (Bad. denitrificans I) reducing the nitrite to
atmospheric nitrogen. 37 In case of associative growth, the aerobic
form removes the free oxygen, thus enabling the other organism to be-
come the denitrifier. Some organisms reduce only nitrates to nitrogen.
The four species found by Maaszen capable of reducing nitrate to gaseous-
nitrogen were Bad. fluorescens liquefaciens, Bad. fluorescens from blood,
Bad. pyocyaneum and Bad. praepollens. These results were confirmed
by other investigators, 39-41 who found Bad. pyocyaneum, Bad. hartlebii
and fluorescent bacteria among the most active denitrifying organisms.

Among the forms capable of reducing nitrates completely to gaseous-
nitrogen, we may also include various organisms isolated from horse
manure, 42 from cattle excreta (Bad. denitrificans agilis) i3 and from the
soil. 44-46 Van Iterson 47 demonstrated the presence in the soil of various
bacteria, namely Bad. stutzeri, Bad. denitrofluorescens and Bad. vul-

38 Zacharowa, T. M. Process of denitrification as dependent upon the reaction
of the medium. Trans. Institute of Fertilizers, No. 15, 1923, Moskau.

39 Sewerin, S. A. Zur Frage fiber die Zersetzung von salpetersauren Salzen
durch Bakterien. Centrbl. Bakt. II, 3: 504-517, 554-563. 1897; 22: 348-370.
1909; 25: 479-492. 1909.

40 Christensen, H. R. Zwei neue fluoreszierende Denitrifikationsbakterien.
Centrbl. Bakt. II, 11: 190-194. 1904.

41 Fred, E. B. Eine physiologische Studie iiber die nitratereduzierenden
Bakterien. Centrbl. Bakt. II, 32: 421-449. 1911.

42 Schirokikh, J. Uber einen neuen Salpeter zerstorenden Bacillus. Centrbl.
Bakt. 11,2: 204-207. 1896.

43 Ampola, G., and Garino, E. Ueber die Denitrifikation. Centrbl. Bakt. II,
2: 670-676. 1896; 3: 309-310. 1897.

44 Jensen, 1897-8 (p. 180).

45 Hoflich, C. Vergleichende Untersuchungen iiber die Denitrifikationsbakter-
ien des Mistes, des Strohes und der Erde. Centrbl. Bakt. II, 8: 245-248, 273-278,
305-308, 336-339, 361-367, 398-406. 1902.

46 Cingolani, M. Recherche intorno al processo della denitrificazione. Staz.
Sper. Agr. ital. 41: 521-538. 1908; Ann. Staz. Chim. Agr. Spes. Roma (2), 2:
274. 1908. (Centrbl. Bakt. II, 23: 238. 1909.)

47 van Iterson, C. Anhaufungsversuche mit denitrifizierenden Bakterien.
Centrbl. Bakt. II, 12: 106-116. 1904.

BACTERIA REDUCING NITRATES AND SULFATES 187

pinus, which reduce nitrates to gaseous nitrogen, in the presence of small
quantities of organic matter. In the same soil, where nitrification takes
place under aerobic conditions, denitrification will take place in the
absence of free oxygen.

The following authentic organisms capable of reducing nitrates to
atmospheric nitrogen have been isolated and described (some of these
are probably only varieties of other species which do not denitrify) :

Bact. denitrificans (= Bact. denitrificans I Burri and Stutzer, Pseud, stutzeri
Mig.) L and N (1.5 to 3 by 0.7/i), a motile, non-spore forming, aerobic organism.

Bact. stutzeri (= Bact. denitrificans II Burri and Stutzer, Bact. nitrogenes Mig.)
L and N (2 to 4 by 0.7 to 0.8ju), a motile, non-spore forming, facultative anaerobic
organism, isolated from straw and horse manure. 48

Bact. kunnemanni (= Bac. denitrificans III Kunnemann), a motile, non-spore
forming organism.

Bact. denitrificans agilis i9 (1 to 1.5 by 0.1 to 0.3/z), a motile, peritrichic, non-
spore forming organism; gram-negative, facultative anaerobic, granulated and
developing slow; according to Lohnis this is a denitrifying variety of Bact. radio-
bacier.

Bact. ulpiani (= Bac. denitrificans VI Ampola et Ulpiani), a motile, non-spore
forming, gram-negative organism.

Vibrio denitrificans** (2 to 4 by 0.5^), a motile, non-spore forming organism.

Bac. schirokikhi, &1 a motile, spore-forming, proteolytic, aerobic organism.

Bact. praepollens,™ a small, non-motile, obligate aerobic organism, reducing
only nitrites.

Bac. nitroxus b3 (No. 62, PI. X) comprising bacilli of variable dimensions,
globous, pyriform, filiform; they take the form of Clostridia at the time of spore
formation, giving an intense glycogen reaction; facultative anaerobic; on
repeated transfer under aerobic conditions may lose faculty of reproduction;
gelatin is liquefied.

In addition to these and the above mentioned bacteria, we may also call
attention to a few other denitrifying forms which were isolated, such as Bact. ful-
vum, bi Bact. hartlebii, hh Bact. centropunctatum, Bact. nitrovorum, B. porticensis, etc.
Most of these organisms are strict aerobes, some being capable of decomposing
proteins actively. Most of them grow on nitrate (0.2 to 1.0 per cent) media, with

48 Kunnemann, O. tJber denitrifizierende Mikroorganismen. Landw. Ver-
suchsta. 50: 65-113. 1898.

49 Ampola and Garino, 1896-1897 (p. 186); Kuntze, VV. Beitriige zur Mor-
phologie und Physiologie der Bakterien. Centrbl. Bakt. II, 13: 1-12. 1904.

"Sewerin, 1897 (p. 186).
61 Jensen, 1898 (p. 180).
62 Maassen, 1899 (p. 181).
63 Beijerinck and Minkman, 1910 (p. 546).

54 Bierema, S. Die Assimilation von Amnion-, Nitrat- und Amidstickstoff
durch Mikroorganismen. Centrbl. Bakt. II, 23: 672-726. 1909.
66 Jensen, 1898 (p. 180).

188 PRINCIPLES OF SOIL MICROBIOLOGY

the formation of a gas (largely N, some C0 2 ) and nitrite. In the absence of free
oxygen, these organisms can exist anaerobically in the presence of nitrate.

A thermophilic denitrifying bacillus (3.5 to 7 by 1 to 1.8/x), facultative
anaerobic, reducing nitrates with the formation of gas and growing at
high temperatures (52°C.) has also been described. 56

Several organisms reducing nitrates are capable of obtaining their
energy from inorganic compounds. Thiob. denitrificans Beij. oxidizes
sulfur and reduces nitrates to nitrogen gas. This organism, or rather
group of organisms, is widely distributed in the soil. 57 - 58 Thiosulfate
can be oxidized by the organism under anaerobic conditions only in
the presence of nitrate as a source of oxygen. 59 The utilization of the
energy obtained by the oxidation of hydrogen gas for the reduction of
nitrates has been pointed out by Niklewski 60 for H. agilis.

The decomposition of cellulose in the soil may be carried on by the
symbiotic action of two bacteria, one reducing nitrate to atmospheric
nitrogen and the other decomposing the cellulose; the decomposition
products of the cellulose are used by the nitrate reducing organism as a
source of energy, which enables it to reduce the nitrate, while the oxygen
thus liberted is utilized by the cellulose decomposing organism, under
anaerobic conditions. 61

Bacteria reducing sulfates to H2S. A detailed study of the formation
of hydrogen sulfide in nature is given elsewhere (p. 600). It is sufficient
to call attention here to the bacteria capable of producing this sub-
stance as a result of reduction of sulfates and other oxygen-rich sulfur
compounds (like thiosulf ates) . Microspira desulfuricans (No. 63, PI.
X), capable of bringing about this reduction, was first studied by
Beijerinck, 62 then obtained in pure culture by Van Delden. 63 It was
isolated on the following medium:

K2HPO4 0.5gram MgS0 4 or CaS0 4 1.0 gram

Sodium lactate 5.0 grams FeSC>4 Trace

Asparagine 1.0 gram Tap water 1000 cc.

88 Ambroz, 1913 (p. 158).
57 Lieske, 1912 (p. 86).
68 Gehring, 1914 (p. 87).
69 Trautwein, 1921 (p. 87).

60 Niklewski, 1914 (p. 99).

61 Gerretsen, 1921 (p. 736).

62 Beijerinck, M. W. tlber Spirillum desulfuricans als Ursache von Sulfatre-
duktion. Centrbl. Bakt. II, 1: 1-9, 48-59, 104-114. 1895.

63 Van Delden, A. Beitrag zur Kenntnis der Sulfatreduktion durch Bak-
terien. Centrbl. Bakt. II, 11: 81-94, 113-118. 1904.

BACTERIA REDUCING NITRATES AND SULFATES 189

The medium is filled to the neck of the bottles, then inoculated
and incubated at 25°C. Sulfur reduction becomes evident by a
change in color due to formation of H 2 S. The bacterium can be
isolated from the soil when some sodium sulfite is added to the medium.
The presence of organic substances as sources of energy and anaerobic
conditions are required for the action of the organism. For the isolation
of pure cultures, 10 per cent gelatin or 2 per cent agar is added to the
above medium; in place of FeS0 4 a trace of FeSO^NH^oSO^GIi^O
together with some sodium carbonate is used. In 3 to 6 days small
black colonies appear. Sulfur is deposited in the colony, on the solid
medium, among the bacterial cells, due to the incomplete reduction of
the sulfate. The organism is a very motile spirillum, 4 by lju in
size and is strictly anaerobic.

Another organism {Microspira aestuarii) was isolated from sea water.
Various thermophilic bacteria (Vibrio thermo desulfuricans) are capable
of reducing sulfates. 04 These three forms are closely related to one
another and have the ability, apart from all other bacteria, to utilize
sulfates and thiosulfates as sources of oxygen under anaerobic conditions.

An actinomyces (A. pelogenes) capable of reducing sulfates to sulfides
and forming iron sulfide was also isolated. 65 These organisms occur in
great abundance in certain lakes and seas, and especially in the black
curative muds; their reducing properties under these conditions keep
the sulfur in the process of constant transformation, 66 as discussed in
detail elsewhere (p. 611).

64 Elion, L. A thermophilic sulfur-reducing bacterium. Centrbl. Bakt. II,
63: 58-67. 1924.

65 Sawyalow, W. tJber Schwefelwasserstoffgiirung im schwarzen Heilsch-
lamme. Centrbl. Bakt. II, 39: 440-447. 1913.

66 Nadson, G. A. On the hydrogen sulfide fermentation in the Weissovo Lake
and the participation of microbes in the formation of the black mud. 1903.
St. Petersburg. (Russian.)

CHAPTER VIII

Bacteria Capable of Decomposing Celluloses and Other

Complex Carbohydrates and Hydrocarbons

in the Soil

Microorganisms concerned in the decomposition of celluloses in nature'
Among the microorganisms concerned in the decomposition of different
constituents of plant and animal tissues, those capable of breaking
down celluloses have attracted considerable attention, due to the fact
that these materials make up a large part of the bulk of the organic
matter added to the soil, but chiefly because the organisms concerned are
more or less specific in nature. Many bacteria are capable of existing
only with celluloses as a source of energy and some cannot even
utilize any other source of energy. Organisms capable of decomposing
celluloses are found among various groups of fungi, among the actino-
myces and among the bacteria. However, under anaerobic conditions,
the fungi and actinomyces do not thrive and bacteria alone are entirely
concerned in the process.

The cellulose-decomposing bacteria can be divided into two groups,
(1) the aerobic and (2) the anaerobic forms. Certain special groups of
these forms may be concerned in the process, namely (3) the ther-
mophilic bacteria, probably active in the decomposition of celluloses
in manure and also in the soil under certain conditions, and (4) the
denitrifying bacteria, active only in the presence of nitrates and under
certain specific conditions. 1

Anaerobic bacteria. Mitscherlich 2 observed in 1850 that, in the rot-
ting of potatoes in water, the cell walls are destroyed while the starch
accumulates at the bottom of the container. He ascribed this action to

1 See Pringsheim, H. Die Polysaccharide. 2 Aufl. Springer. Berlin. 1923;
Karrer, P. Einfiihrung in die Chemie der polymeren Kohlenhydrate. Akad.
Verlagsges. Leipzig. 1926. Rippel, A. Der biologische Abbau der pflanz-,
lichen Zellmembrannen. Ztschr. angew. Bot. 1: 78-97. 1919; Waksman, S. A.
and Skinner, C. E. The microorganisms concerned in the decomposition of
celluloses in the soil. Jour. Bact. 12: 57-84. 1926.

2 Mitscherlich. Zusammensetzung der Wand der Pflanzenzelle. Monatschr.
K. Akad. Wiss. Berlin. 1850, 102-110.

190

BACTERIA DECOMPOSING CELLULOSES 191

vibrios which were present in abundance in the water. Van Tieghem 3
described in detail a species of Amylobacter previously found to occur
in decomposing plant tissues and staining blue with iodine; it decom-
posed young plant tissues with the formation of butyric acid, carbon
dioxide and hydrogen. However, this organism was not a species in
the true sense of the word, but a collective form; it is doubtful whether
it decomposed pure cellulose at all, so that it could hardly deserve the
term "cellulose organism." 4 Since cellulose forms an important con-
stituent of manure, attention has been directed chiefly towards cellulose
decomposition in the rotting of manure. It has been found, for ex-
ample that the atmosphere at different depths of the manure pile con-
sists of various gases. The content of carbon dioxide and especially of
methane increases and the nitrogen content decreases with depth.
Oxygen is entirely absent at the lower depths of the pile.

Omeliansky 5 was the first to establish definitely the connection be-
tween the activities of microorganisms and the decomposition of
cellulose.

The following medium was employed:

K 2 HP0 4 l.Ogram NaCl trace

MgS0 4 0.5 gram Distilled water 1000 cc.

(NH 4 ) 2 S0 4 or\

(NH 4 ) 2 HP0 4 f • 10gram

The ammonium salt may be replaced by 0.5 per cent asparagine or 0.1 per cent
peptone. Some chalk and pure filter paper were placed in long-necked bottles,
which were then filled with the medium to the stopper. The flasks were inocu-
lated with horse manure or river mud and incubated at 34° to 35°. After a con-
siderable period of incubation (usually more than a week), gas production set in.
The